Figure 2.

5A6 induces direct cytotoxicity, ADCC, CDC, and ADCP and is unique among anti-CD81 mAbs. (A and B) 10⁶ Raji cells were incubated overnight with 1 µg/ml of mouse anti-CD81 (5A6), rituximab, or different anti-CD81 mAbs clones (5A6, 1D6, JS81, and 1.3.3.22, all MsIgG1 isotypes) in the absence (A) or presence (B) of purified human NK cells (5:1). Cell death was measured by Annexin-V and 7-AAD staining. Ctrl, control. (C) 10⁶ Raji cells were incubated with 10 µg/ml of 5A6 (MsIgG1, MsIgG2a, or chimeric HuIgG1), rituximab, or control antibody for 1.5 h in the presence of fresh pooled human serum. (D) Raji cells labeled with pHrodo green AM (fluorescence increase in acidic pH denotes active phagocytosis) were opsonized for 15 min with the indicated mAbs. Cells were then incubated for 4 h at a 1:3 ratio with peritoneal mouse macrophages previously activated with 100 ng/ml of LPS for 24 h. Phagocytic events were defined as percentage of F4/80 and high MFI pHrodo green AM double-positive macrophages. All experiments were done in triplicate in at least two independent experiments. Error bars represent mean ± SEM. *, P < 0.0149; **, P < 0.0015; ***, P < 0.0001; Student’s t test (A–D).