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. 2008 May 1;111(9):4813-6.
doi: 10.1182/blood-2008-01-133785. Epub 2008 Mar 7.

Simultaneously targeting CD45 significantly increases cytotoxicity of the anti-CD33 immunoconjugate, gemtuzumab ozogamicin, against acute myeloid leukemia (AML) cells and improves survival of mice bearing human AML xenografts

Roland B Walter  1 , Kelli M Boyle, Frederick R Appelbaum, Irwin D Bernstein, John M Pagel
Affiliations

Simultaneously targeting CD45 significantly increases cytotoxicity of the anti-CD33 immunoconjugate, gemtuzumab ozogamicin, against acute myeloid leukemia (AML) cells and improves survival of mice bearing human AML xenografts

Roland B Walter et al. Blood. .

Abstract

Targeting CD33 or CD45 is currently exploited for immunotherapy of acute myeloid leukemia (AML). Gemtuzumab ozogamicin (GO), an immunoconjugate of an anti-CD33 antibody that facilitates cellular uptake of a toxic calicheamicin-gamma(1) derivative, induces complete remissions in a subset of patients with AML. We herein tested whether simultaneous targeting of CD45 could improve GO cytotoxicity against AML cell lines and primary AML cells. We found that the anti-CD45 antibody, BC8, dose-dependently increased cytotoxicity induced by GO, and, to a lesser degree, free calicheamicin-gamma(1). BC8 promoted CD33 endocytosis, suggesting that its effect on GO cytotoxicity may be, at least partly, due to increased uptake and intracellular GO availability. Finally, compared with either agent alone, BC8 combined with GO resulted in marked tumor growth inhibition and superior survival rates of mice bearing human AML xenografts. These data suggest that further study of this antibody combination for clinical use in AML is warranted.

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Figures

Figure 1
Figure 1
Effect of BC8 on GO- and calicheamicin-γ1–induced cytotoxicity as well as CD33 internalization and modulation in human AML cell lines in vitro. (A,B) Drug-induced cytotoxicity. ML-1 and HL-60 cells (top and bottom panels, respectively) were incubated with various concentrations of (A) GO or (B) calicheamicin-γ1 for 3 days in the presence or absence of increasing concentrations of BC8. Cytotoxicity was assessed using PI staining and expressed as the percentage of PI+ cells. (C) GO-induced cytotoxicity in presence of BC8 and 31A. ML-1 cells were incubated with various concentrations of GO for 3 days in the presence or absence of BC8 (20 μg/mL) and/or 31A (20 μg/mL). (D) CD33 endocytosis. ML-1 and HL-60 cells (top and bottom panels, respectively) were incubated for 30 minutes with medium containing 2.5 μg/mL unconjugated, unlabeled hP67.6 in ice water to prevent internalization during the staining procedure. Cells were then washed in ice-cold PBS to remove unbound antibody, resuspended in antibody-free medium, and incubated at 37°C (in 5% CO2 and air) for various periods of time. Subsequently, cells were chilled and incubated with biotin-conjugated mouse anti–human IgG4 monoclonal antibody (5 μg/mL), followed by incubation with streptavidin-PE conjugate (5 μg/mL) to detect remaining hP67.6 on the cell surface. One sample that was kept in ice water was used to determine the starting level of antibody bound to the cell. (E) CD33 modulation. ML-1 and HL-60 cells (top and bottom panels, respectively) were incubated overnight in the presence or absence of hP67.6 and/or BC8. Cell surface CD33 was then measured by subsequent staining with hP67.6, a biotin-conjugated mouse anti–human IgG4 antibody, and a streptavidin-PE conjugate. Results are expressed as arbitrary fluorescence units (AFU) and shown as mean plus or minus SEM from 3 to 7 independent experiments. *P < .05; #P < .01; $P < .001.
Figure 2
Figure 2
In vivo effect of BC8 on GO-induced cytotoxicity in mice bearing human AML xenografts. (A) Tumor volumes. Athymic mice bearing HL-60 xenografts were randomized and either left untreated (●) or injected intraperitoneally with BC8 (10 μg, ■) on a 5 days on/2 days off schedule for 5 cycles, GO (5 μg, ▴) on days +1, +5, +9, +22, +26, and +30, or BC8 (10 μg) combined with GO (5 μg) (▾) administered on an identical schedule as in the individual treatment group. Tumors were measured in 3 dimensions with calipers 3 times per week. Tumor volumes of dead mice were maintained in the curve until the last mouse of the corresponding group was killed. Results are shown as mean plus or minus SEM. CR denotes complete resolution of measurable tumor. *P < .05 compared with treatment with BC8 alone. (B) Survival. Groups of 10 mice bearing HL-60 tumor xenografts were either left untreated (●) or injected intraperitoneally with BC8 (10 μg, ■), GO (5 μg, ▴), or a combined therapy of BC8 (10 μg) and GO (5 μg) (▾) as described for panel A, and analyzed for survival as a function of time. Proportions of surviving animals in each treatment group are shown. Mice were killed if the tumors caused discomfort, impaired ambulation, or weight loss of more than 30% of starting weight. Four of 10 animals survived after combined therapy with BC8 and GO, whereas none of the animals treated with either BC8 or GO survived (P = .008; Fisher exact test); similarly, all untreated animals died.
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