Figure 5.
Characterization of ChT-1A5 and its induction of antibody-dependent cellular cytotoxicity in AML cell lines and primary cells. (A) Binding kinetics of anti-B7–H3 mAbs T-1A5 and ChT-1A5 to full B7-H3 protein measured by surface plasmon resonance (Octet). (B) Timelapse fluorescence data show apoptosis induced by NK-cell ADCC. U937 cells were treated with ChT-1A5 or rituximab at 1, 5, and 10 μg/mL in the presence of NK cells at an 8:1 NK:target ratio. Apoptosis was measured every hour for 30 hours. Leukemic cells were labeled with CytoLight red and apoptotic cells with annexin V (green). (C) Bar graph showing the percentage of annexin V binding at 28 hours in U937 cells treated with ChT-1A5 or rituximab at 1, 5, and 10 μg/mL in the presence of NK cells in an 8:1 NK:target ratio. Data are plotted as mean values with error bars representing standard error (Student unpaired t test). (D) Cell death kinetics represents induction of apoptosis in primary AML cells (3 patients) treated with ChT-1A5 or rituximab at 1 μg/mL in the presence of NK cells measured every hour for 12 hours for 2 patients and 36 hours in the third patient, at a 4:1 NK:target ratio. (E) Bar graphs (mean values with standard error bars) represent the percentage of cell death in primary AML cells treated with ChT-1A5 or rituximab at 1 μg/mL in the presence or absence of NK cells at a 4:1 NK:target ratio at 12, 12, and 36 hours, from left to right. (Student unpaired t test). (F) Cell death kinetics and bar diagram (G) represent induction of apoptosis in PBMCs derived from healthy donors (Control-1). PBMCs were treated with ChT1-A5 mAb or rituximab at 1 μg/mL in the presence or absence of NK cells (8:1 effector:target ratio); PBMCs were labeled with CytoLight red, and annexin V. (H) Bar graph (mean values with standard error bars) represent the percentage of cell death in primary AML cells treated with ChT-1A5/T-1A5 or combination of ChT1-A5 and T-1A5 at 1 μg/mL in the presence or absence of NK cells at a 2:1 NK:target ratio at 76 hours (Student unpaired t test). NS not significant; Rx, rituximab; Scr, scrambled. ***P < .0001.

Characterization of ChT-1A5 and its induction of antibody-dependent cellular cytotoxicity in AML cell lines and primary cells. (A) Binding kinetics of anti-B7–H3 mAbs T-1A5 and ChT-1A5 to full B7-H3 protein measured by surface plasmon resonance (Octet). (B) Timelapse fluorescence data show apoptosis induced by NK-cell ADCC. U937 cells were treated with ChT-1A5 or rituximab at 1, 5, and 10 μg/mL in the presence of NK cells at an 8:1 NK:target ratio. Apoptosis was measured every hour for 30 hours. Leukemic cells were labeled with CytoLight red and apoptotic cells with annexin V (green). (C) Bar graph showing the percentage of annexin V binding at 28 hours in U937 cells treated with ChT-1A5 or rituximab at 1, 5, and 10 μg/mL in the presence of NK cells in an 8:1 NK:target ratio. Data are plotted as mean values with error bars representing standard error (Student unpaired t test). (D) Cell death kinetics represents induction of apoptosis in primary AML cells (3 patients) treated with ChT-1A5 or rituximab at 1 μg/mL in the presence of NK cells measured every hour for 12 hours for 2 patients and 36 hours in the third patient, at a 4:1 NK:target ratio. (E) Bar graphs (mean values with standard error bars) represent the percentage of cell death in primary AML cells treated with ChT-1A5 or rituximab at 1 μg/mL in the presence or absence of NK cells at a 4:1 NK:target ratio at 12, 12, and 36 hours, from left to right. (Student unpaired t test). (F) Cell death kinetics and bar diagram (G) represent induction of apoptosis in PBMCs derived from healthy donors (Control-1). PBMCs were treated with ChT1-A5 mAb or rituximab at 1 μg/mL in the presence or absence of NK cells (8:1 effector:target ratio); PBMCs were labeled with CytoLight red, and annexin V. (H) Bar graph (mean values with standard error bars) represent the percentage of cell death in primary AML cells treated with ChT-1A5/T-1A5 or combination of ChT1-A5 and T-1A5 at 1 μg/mL in the presence or absence of NK cells at a 2:1 NK:target ratio at 76 hours (Student unpaired t test). NS not significant; Rx, rituximab; Scr, scrambled. ***P < .0001.

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