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Blood, Vol. 111, Issue 4, 2211-2219, February 15, 2008
Bispecific anti-CD20/22 antibodies inhibit B-cell lymphoma proliferation by a unique mechanism of action
Blood Qu et al.
111: 2211
Supplemental materials for: Qu et al
Files in this Data Supplement:
- Figure S1. Binding of the recombinant anti-CD20/22 bsAbs to CD22 (JPG, 36.4 KB)
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The binding specificity and affinity of the scFv moieties of hA20IgG-(LL2scFv)2 and hA20IgG-(RFB4scFv)2 were evaluated by competitive binding against HRP-LL2 and biotin-RFB4, respectively. Measurements were performed in duplicate and error bars are shown. (A) Blocking of HRP-LL2 binding by hA20IgG-(LL2scFv)2 (▲), hA20Fab-LL2scFv (■), or epratuzumab (●). (B) Blocking of biotin-RFB4 binding by hA20IgG(RFB4scFv)2 (▲), hA20FabRFB4scFv (■), or cRFB4 (●). The difference between the IgG-scFv2 and Fab-scFv pair is not statistically significant, P > 0.4, while the difference between an antiCD20/22 bsAb and the parental anti-CD22 mAb is significant, P < 0.0001.
- Figure S2. Localization of cell surface bound anti-CD20/22 bsAbs (JPG, 47.6 KB)
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Ramos cells were incubated in medium containing 10 µg/ml of hA20IgG-(RFB4scFv)2 or cRFB4 (indicated on top) on ice for 1 h. After washing to remove unbound Abs, the cells were resuspended in fresh medium, split into two portions, and incubated for 30 min at 4 or 37°C, respectively (indicated at left side). The cells were then treated with cold Formald-Fresh solution and stained with FITC-goat anti-human (GAH) IgG, Fc fragment specific Ab. After washing, the cells were visualized with Olympus BX60 fluorescence microscope and the images were captured with Kodak MDS290 system.
- Figure S3. Membrane localization of CD20 in Daudi cells (JPG, 37.8 KB)
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The cells were treated for 15 min with 100 nM of hA20IgG-(LL2scFv)2, epratuzumab, hA20 (veltuzumab), TF3, or  -IgM, then lysed in a buffer containing 0.5% CHAPS. The lysates were fractionated by sucrose density-gradient ultracentrifugation. The presence of CD20 in the soluble fractions and lipid rafts was probed by Western blots using a rabbit CD20-specific Ab. The positions of CD20 are indicated on right. Lanes of samples treated with hA20IgG-(LL2scFv)2 and TF3 are labeled as -CD20/22-L and -CD20/22-DNL, respectively.
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