CD37-SMIP induced direct cytotoxicity and ADCC in human B-cell lines. (A) CD37-SMIP induced direct cytotoxicity in CD37⁺ (Raji and Ramos), but not in CD37⁻(697) human B-cell lines. Cytotoxicity was measured using FITC-Annexin V/PI staining at 24 hours after treatment as described in the legend to Figure 1A. Representative results from 3 independent experiments are shown. Error bars are SD among triplicate samples within the same experiment. (B) CD37-SMIP induced direct cytotoxicity in Raji B-cell lines comparable with that seen with alemtuzumab or rituximab. In vitro direct cytotoxic effects of CD37-SMIP, alemtuzumab, or rituximab in the presence or absence of cross-linker (α-Fc) was evaluated by FITC-Annexin V/PI staining. Percentage cytotoxicity was measured using FITC-annexin V/PI staining at 24 hours after treatment as described above. Raji parent clone (CD52⁻) and variant CD52⁺ clones were used in these studies. (C) CD37-SMIP induced ADCC in CD37⁺ Raji cells but not in CD37⁻ 697 B-cell line. Effect of PBMC from healthy donors to mediate trastuzumab-, CD37-SMIP-, alemtuzumab-, or rituximab-dependent ADCC function against CD37⁺ (Raji cells) or CD37⁻ (697 cells) target cells was evaluated at the indicated effector: target (E:T) ratios by standard chromium release assay as described in legend to Figure 3A. (D) Rituximab but not CD37-SMIP mediated CDC function against Raji B-cell line. Raji cells were treated with media, trastuzumab, CD37-SMIP, rituximab, or alemtuzumab in the presence of media, human plasma, or heat-inactivated human plasma for 1 hour. The CDC function was evaluated by propidium iodide staining and presented as percentage of PI positive cells in response to the various treatments. Results shown are representative of three independent experiments.