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CD20 and CD37 antibodies show synergy in CDC
Next, the effect of the Fc-Fc interaction-inhibiting muta- tions on colocalization of Hx-CD20-11B8 and Hx-CD37 mAbs on the cell membrane of Daudi cells was evaluated using FRET analysis. mAb combinations with Hx-CD20- 11B8 and Hx-CD37 variants, both harboring the same Fc- Fc inhibiting mutation (K439E or S440K) showed reduced FRET on Daudi cells (Figure 5G and Online Supplementary Figure S6). FRET levels were restored when Fc-Fc inhibi- tion was neutralized by combining Hx-CD20-11B8 and Hx-CD37 mAbs, each having one of the complementary mutations K439E or S440K. Thus, donor- and acceptor- labeled Hx-CD20-11B8 and Hx-CD37 mAb variants come together in close proximity on the cell membrane of Daudi cells, which appears to be, at least in part, mediated by the Fc domain.
Combinations of hexamerization-enhanced CD20 and CD37 mAbs induce superior ex vivo complement-dependent cytotoxicity of tumor cells obtained from patients with B-cell malignancies
We next examined the capacity of combinations of Hx- CD20 and Hx-CD37 mAbs to induce CDC ex vivo in tumor cells obtained from patients with B-cell malignan- cies. First, the CDC activity of Hx-CD20-7D8, Hx-CD37 or combinations was evaluated using tumor cells obtained from 15 patients diagnosed with CLL. Both Hx-CD20- 7D8 and Hx-CD37 induced substantial CDC of CLL tumor cells from all 15 tested donors (Figure 6A), in accor- dance with that seen in Figure 1. Hx-CD37 was more effective in CDC than Hx-CD20-7D8, which may be explained by higher expression CD37 on CLL cells
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B
CD
Figure 6. Combinations of hexamerization-enhanced CD20 and CD37 monoclonal antibodies (mAbs) induce superior ex vivo complement-dependent cytotoxicity (CDC) of tumor cells obtained from patients with B-cell malignancies. (A) B cells obtained from 15 patients diagnosed with chronic lymphocytic leukemia (CLL) were opsonized with fixed concentrations of hexamerization-enhanced type I CD20 mAb 7D8-derived Hx-CD20-7D8 or hexamerization-enhanced CD37 mAb 37.3-derived Hx-CD37 (open symbols: 2.5 μg/mL, closed symbols: 2 μg/mL; each presented as 100%), or 1:1 mixtures thereof (open symbols: 0.625 μg/mL of each mAb, closed symbols: 0.5 μg/mL of each mAb; each represented as 50%) in the presence of 50% NHS. CDC induction is presented as the percentage lysis determined by the fraction of TO-PRO-3 positive cells. (B) B cells of a representative CLL patient sample (patient G) were opsonized with different total mAb concentrations of Hx-CD20- 7D8 or Hx-CD37 (single agents indicated as 100%) and combinations thereof at different antibody ratios (indicated as 75%:25%, 50%:50% and 25%:75%) in the presence of 50% NHS. CDC induction is presented as the percentage lysis determined by the fraction of TO-PRO-3 positive cells. Data shown are mean and Standard Deviation of duplicate measurements. (C) B cells obtained from ten patients diagnosed with different B-cell malignancies [B-cell non-Hodgkin lymphoma (B-NHL) not otherwise specified (NOS), follicular lymphoma (FL), marginal zone lymphoma (MZL) and mantle cell lymphoma (MCL)] were opsonized with 10 μg/mL of hexamer- ization-enhanced type II CD20 mAb 11B8-derived Hx-CD20-11B8 or Hx-CD37, and the combination thereof (5 + 5 μg/mL) in the presence of 20% NHS. CDC induction is presented as the percentage lysis determined by the fraction of 7-AAD positive B-lymphoma cells. (D) CDC assay with B-cell patient samples representative for B- NHL (NOS), FL, MZL and MCL as described in (C). Data shown are mean and Standard Deviation of duplicate measurements. *P<0.05; **P<0.01; ****P<0.0001.
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