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CD20 and CD37 antibodies show synergy in CDC
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Figure 2. CD20 and CD37 mAbs synergistically induce complement-dependent cytotoxicity (CDC) of malignant B cells. (A and B) CDC on Daudi cells opsonized with 30 μg/mL wild-type (WT) type I CD20 mAb 7D8 (IgG1-CD20-7D8) (A) or type II CD20 mAb 11B8 (IgG1-CD20-11B8) (B), CD37 mAb 37.3 (IgG1-CD37), or a combination thereof (15 + 15 μg/mL) in the presence of 20% NHS. CDC induction is expressed as the percentage lysis determined by the fraction of propidium iodide (PI)-positive cells. Data shown are mean and Standard Deviation of triplicate measurements. (C) 8 x 8 CDC dose response matrix plot for the combination of hexamerization- enhanced CD37 mAb Hx-CD37 (0-0.8 μg/mL) with hexamerization-enhanced CD20 mAb Hx-CD20-11B8 (0-8 μg/mL), tested on Daudi cells and categorized as a color gradient from green (0% lysis) to yellow (50% lysis) to red (100% lysis). HIV gp120-specific mAb b12 (IgG1-gp120) was used as a negative control human mAb. (D) Loewe additivity-based combination index (CI) values calculated by CompuSyn for the CDC dose response matrix as described in (C) and categorized as synergistic (<1, red), additive (1, white) and antagonistic (>1, blue). Representative examples of two replicate experiments are shown. (E) CDC and CD37 expression analysis on Daudi, Raji and WIL2-S cells. For the CDC assay, cells were opsonized with Hx-CD37 (10 μg/mL), different CD20 mAb variants (10 μg/mL) or combinations thereof (10 + 10 μg/mL). Data show the mean of nine replicates collected from three independent experiments. Expression levels were determined using QIFIKIT analysis. The number of antibody molecules per cell was calculated from the antibody-binding capacity (mean fluorescence intensity, MFI) normalized to a calibration curve, according to the manufacturer’s guidelines. Expression data show the mean of four replicates collected from two independent experiments. ****P<0.0001.
haematologica | 2019; 104(9)
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