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S.C. Oostindie et al.
Combinations of Hx-CD20-7D8 and Hx-CD37 did not significantly increase C1q binding. However, the combi- nation showed higher CDC efficacy as demonstrated by the lower EC50 value in the C1q dose-response curves compared to the single mAbs (0.03 μg/mL for the combi- nation vs. 0.12 μg/mL for Hx-CD20-7D8 and 0.34 μg/mL for Hx-CD20-11B8) (Figure 3B). In contrast to the results with the type I CD20 mAb-derived variant, combining type II CD20 mAb-derived Hx-CD20-11B8 with Hx- CD37 resulted in increased C1q binding compared to the single mAbs, as well as increased CDC efficacy (Figure 3C and D). Collectively, these data suggest that combinations of both type I and type II CD20 mAb-derived Hx-CD20 mAb with Hx-CD37 mAbs activate complement more effectively.
CD20 and CD37 mAbs colocalize on B cells
Confocal microscopy was used to determine whether the CD20- and CD37-specific antibodies associate on the cell surface upon target binding. Cell-bound Hx variants of CD20 mAb 7D8 and CD37 mAb 37.3 were detected using A488 and A594 fluorescent labeling, respectively, and anti- body colocalization was quantified by calculating spatial overlap (Manders’ co-efficients) between the two fluores- cent labels. The merged A488/A594 image showed that membrane-bound Hx-CD20 and Hx-CD37 mAbs indeed colocalized on the surface of Raji cells (Figure 4A), which was confirmed by quantitative analysis, giving Manders' coefficients of M1=0.805 (fraction of image 1 overlapping image 2) and M2=0.751 (fraction of image 2 overlapping image 1).
AB
CD
E
Figure 1. Hexamerization-enhancing mutations in CD20 and CD37 mAbs sub- stantially enhance complement-dependent cytotoxicity (CDC) of chronic lym- phocytic leukemia (CLL) B cells. (A and B) CDC of B cells obtained from patient A with CLL. Cells were opsonized with different concentrations of CD20 mAb 7D8 as wild type (IgG1-CD20-7D8) or with a hexamerization-enhancing muta- tion (Hx-CD20-7D8) (A); or CD37 mAb 37.3 as wild type (IgG1-CD37) or with a hexamerization-enhancing mutation (Hx-CD37) (B) in the presence of 50% pooled normal human serum (NHS), heat-inactivated (HI) NHS, NHS + EDTA or medium. Representative examples of three replicate experiments are shown. (C-E) CDC of B cells obtained from 12 different CLL patients (patient B-M). CLL B cells were opsonized with 16 μg/mL (C), 2 μg/mL (D) or 0.25 μg/mL (E) Hx- CD20-7D8 or Hx-CD37. The dashed line represents 95% cell lysis. CDC induc- tion is expressed as the percentage lysis determined by the fraction of TO-PRO- 3 positive cells and data shown are mean and Standard Deviation of duplicate measurements.
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haematologica | 2019; 104(9)