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Blood, Vol. 110, Issue 12, 3959-3967, December 1, 2007
Anti-BR3 antibodies: a new class of B-cell immunotherapy combining cellular depletion and survival blockade
Blood Lin et al.
110: 3959
Supplemental materials for: Lin et al
Files in this Data Supplement:
- Figure S1. B cell reduction in response to different doses of anti-BR3 mAb (JPG, 49.2 KB)
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Mice were treated with 200, 20, 2 and 0.2 µg anti-BR3 antibody and blood and spleen (follicular and marginal zone) B cells were enumerated at day 4 as described in figure 1.
- Figure S2. Kinetics of B cell reduction in response to anti-BR3 mAb and anti-CD20 mAb treatment (JPG, 68 KB)
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In both blood (1 hour after treatment) and lymph nodes (1 day) anti-CD20 depletes B cells faster than anti-BR3. By 8 days of treatment both mAbs deplete B cells comparably in blood.
- Figure S3. Mouse and human BR3 surface levels on B cells and plasma cells (JPG, 73.7 KB)
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BR3 shows high surface expression on B cells, and reduced but measurable expression on spleen and bone marrow plasma cells.
- Figure S4. Anti-BR3 mAb and BR3-Fc treatment results in B cell reduction in the NZB/W F1 strain (JPG, 102 KB)
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After 6 weeks of treatment with anti-BR3 mAb or BR3-Fc, B cells (B220+) are reduced in NZB/W F1 mice, but not to the same extent as in non-autoimmune mouse strains (compare this data with figures 1 and 2). C57BL/6 mice were included as control in one experiment side by side with NZB/W F1 mice and showed B cell reduction comparable to that shown in Figure 2 (data not shown).
- Figure S5. Anti-BR3 mAb and BR3-Fc reduce both cycling and non-cycling B cells, plasma cells and plasmablasts while cytoxan reduces primarily cycling cells (JPG, 92.4 KB)
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After one week of treatment with control mAb, anti-BR3 mAb or BR3-Fc, BrdU positive and negative B cells (B220+) or plasma cells (CD138+) were quantified by flow cytometry (top – plots of representative animals from each group). Cycling (BrdU+) and non-cycling (BrdU−) total B cells, IgM+ and IgG2a+ plasma cells decrease proportionally in anti-BR3 mAb and BR3-Fc treated groups while cytoxan reduces primarily cycling cells and increases the ratio of BrdU−/BrdU+ remaining cells (bottom).
- Figure S6. CLL B cells with downregulated CD20 express BR3 (JPG, 57.8 KB)
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Control and CLL PBMCs were stained for CD19 and CD5 to identify normal (CD19+CD5−/lo – non-maligant) and malignant (CD19+CD5hi) B cells. Most CLL patients have low (CLL 2, 4, 5, 10, 11, 12, 13, 15) to negligible (CLL 1, 3, 9) levels of CD20 on the malignant B cells compared with residual non-malignant cells and B cells from normal donors. In most patients, BR3 levels show less reduction than CD20. Sample MFIs (for BR3 and CD20 respectively) were calculated as percent of the average of 4 normal donor B cells (normal B).
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