Figure 4.
Figure 4. CD19-DE efficacy is dependent on the presence of macrophages in vivo and effectively mediates phagocytosis of ALL cells in vitro. (A) NSG mice were treated with a single injection of liposomal clodronate intraperitoneally, and their bone marrow was analyzed for macrophage depletion by flow cytometry 6 days later. (B) Patient-derived ALL xenografts were established by orthotopic intrafemoral transplantation of 1000 ALL cells per animal in NSG mice (patient 1; day 0). Mice were treated with CD19-DE, as is depicted in Figure 1B (“antibody only”), with LC alone on day −1 and every 7 days thereafter (“LC only”) or with a combination of CD19-DE and LC (“antibody + LC”). Postmortem analyses of the mice that did not succumb to LC toxicity: splenic volume (left) as a marker of leukemic burden; human leukemic blasts in the bone marrow (middle); and human leukemic blasts in the spleen in a subset of mice (right) (Mann-Whitney U test). Mice that could be evaluated for spleen volume and bone marrow infiltration on day +65: control n = 20; LC only n = 16; antibody n = 22; and LC + antibody n = 13. Mice that could be evaluated for spleen blasts on day +65: control n = 8; LC only n = 8; antibody n = 8; and LC + antibody n = 6. (C) Microscopy analyses of antibody-mediated phagocytosis of green-fluorescent 697 ALL cells by human macrophages. Phagocytosis of a leukemic cell by a macrophage (arrows). (D) Enhancement of antibody-mediated phagocytosis of 697 ALL cells by treatment with antibody CD19-DE in vitro (each dot represents an independent experiment, ANOVA, and Bonferroni’s multiple-comparison test). (E) Enhancement of antibody-mediated phagocytosis of 4 primary xenograft-derived patient ALL cells (patients 1-4 in Table 1) by treatment with antibody CD19-DE in vitro (each symbol represents an independent patient, ANOVA, and Bonferroni’s multiple comparison test). Replicate analyses of the individual patients are depicted in Supplemental Figure 3. A, ALL cell; CD19 IgG1, CD19-specific control antibody not containing the CD19-DE Fc modification; IgG1, nontargeting control antibody; i.p., intraperitoneal; lipo-clod, liposomal clodronate; M, macrophage. *P < .05. **P < .01. ***P < .001.

CD19-DE efficacy is dependent on the presence of macrophages in vivo and effectively mediates phagocytosis of ALL cells in vitro. (A) NSG mice were treated with a single injection of liposomal clodronate intraperitoneally, and their bone marrow was analyzed for macrophage depletion by flow cytometry 6 days later. (B) Patient-derived ALL xenografts were established by orthotopic intrafemoral transplantation of 1000 ALL cells per animal in NSG mice (patient 1; day 0). Mice were treated with CD19-DE, as is depicted in Figure 1B (“antibody only”), with LC alone on day −1 and every 7 days thereafter (“LC only”) or with a combination of CD19-DE and LC (“antibody + LC”). Postmortem analyses of the mice that did not succumb to LC toxicity: splenic volume (left) as a marker of leukemic burden; human leukemic blasts in the bone marrow (middle); and human leukemic blasts in the spleen in a subset of mice (right) (Mann-Whitney U test). Mice that could be evaluated for spleen volume and bone marrow infiltration on day +65: control n = 20; LC only n = 16; antibody n = 22; and LC + antibody n = 13. Mice that could be evaluated for spleen blasts on day +65: control n = 8; LC only n = 8; antibody n = 8; and LC + antibody n = 6. (C) Microscopy analyses of antibody-mediated phagocytosis of green-fluorescent 697 ALL cells by human macrophages. Phagocytosis of a leukemic cell by a macrophage (arrows). (D) Enhancement of antibody-mediated phagocytosis of 697 ALL cells by treatment with antibody CD19-DE in vitro (each dot represents an independent experiment, ANOVA, and Bonferroni’s multiple-comparison test). (E) Enhancement of antibody-mediated phagocytosis of 4 primary xenograft-derived patient ALL cells (patients 1-4 in Table 1) by treatment with antibody CD19-DE in vitro (each symbol represents an independent patient, ANOVA, and Bonferroni’s multiple comparison test). Replicate analyses of the individual patients are depicted in Supplemental Figure 3. A, ALL cell; CD19 IgG1, CD19-specific control antibody not containing the CD19-DE Fc modification; IgG1, nontargeting control antibody; i.p., intraperitoneal; lipo-clod, liposomal clodronate; M, macrophage. *P < .05. **P < .01. ***P < .001.

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