BC133 has distinct binding properties to CD33 and CD3. (A) MOLM13 cells were reacted for 30 minutes at 4°C using decreasing doses of hM195 (anti-CD33 IgG), the heterodimeric BsAb, and BC133. Cells were washed and immunostained with a fluorochrome-conjugated secondary antibody and mean fluorescence intensity (MFI) was assayed by flow cytometry. (B) MOLM13 cells were reacted for 30 minutes, 4 hours, or 24 hours at 37°C or 4°C with 1 μg/mL of huM195 (anti-CD33 IgG), the heterodimeric IgG BsAb, and BC133. Cells were washed and immunostained with a fluorochrome-conjugated secondary antibody at 4°C. After washing the unbound antibody, MFI was analyzed by flow cytometry. (C) Activated T cells were reacted for 30 minutes at 4°C with decreasing doses of huOKT3 (anti-CD3 IgG), the heterodimeric BsAb, and BC133. Cells were washed and immunostained with a fluorochrome-conjugated secondary antibody, and MFI was measured by flow cytometry.