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CD20 and CD37 antibodies show synergy in CDC
between two groups were analyzed using paired Student t-test with two-tailed 95% Confidence Intervals and between more groups by paired or unpaired one-way ANOVA followed by a Tukey’s post-hoc multiple comparisons test. See Online Supplementary Methods for details on synergy and colocalization analysis.
Results
Hexamerization-enhancing mutations in CD20 and CD37 mAbs substantially enhance complement-dependent cytotoxicity of chronic lymphocytic leukemia B cells
We previously reported increased CDC with engineered mAbs containing Hx mutations in the Fc domain.15,16 We therefore investigated whether introducing the Hx mutation E430G into the CD37 chimeric IgG1 mAb 37.3 could potentiate CDC in B cells isolated from chronic lymphocytic leukemia (CLL) patients and compared this to the CD20 mAb IgG1-CD20-7D8 with and without a Hx mutation. Wild-type (WT) IgG1-CD20-7D8 promoted considerable CDC of CLL B cells and CDC was increased by the E430G mutation (Figure 1A). While WT IgG1-CD37 efficiently binds to CLL B cells, it was ineffec- tive at inducing CDC (Figure 1B and Online Supplementary Figure S2), in contrast to Hx-CD37 (Figure 1B). For both Hx-CD20-7D8 and Hx-CD37, high levels of cell killing largely required active complement, since CDC was almost absent in heat-inactivated NHS, NHS supplement- ed with EDTA or medium alone (Figure 1A and B). Background killing of cells from patient A mediated by Hx-CD37 in the absence of complement, was slightly higher than expected. However, in C1q-depleted serum, background killing was 16%, compared to 6% for cells reacted with Hx-CD20-7D8. Background killing in C1q- depleted serum for six other CLL patient samples aver- aged 13% and 14% for cells reacted with Hx-CD20-7D8 and Hx-CD37, respectively. Reaction in NHS increased CDC to averages of 91% and 95%, respectively. Introduction of the Hx mutation E430G into CD20 and CD37 mAbs did not affect pharmacokinetic profiles and binding to FcRn (data not shown).16 At the highest concen- tration (16 μg/mL) Hx-CD37 induced ≥95% CDC of tumor B cells for 9 of 12 patients (Figure 1C). At concen- trations of 0.25 and 2 μg/mL, Hx-CD37 generally demon- strated higher potency than Hx-CD20-7D8 (Figure 1D and E), which may be explained by higher expression levels of CD37 (approximately 2-fold) in the majority of CLL sam- ples (Online Supplementary Figure S3A and B).
CD20 and CD37 mAbs synergistically induce complement-dependent cytotoxicity of malignant B cells
We investigated the CDC activity of combinations of WT CD20 and WT CD37 mAbs using two different CD20 mAbs. The ability to activate complement represents a key distinction between type I CD20 mAbs, which medi- ate strong CDC, and type II CD20 mAbs, which only mediate weak CDC.32 The effect of combining WT type I CD20 mAb 7D8 or WT type II CD20 mAb 11B8 with WT CD37 mAbs on CDC was assessed using Daudi cells. As expected, WT IgG1-CD20-7D8 showed potent CDC activity (96.6% cell lysis), whereas WT IgG1-CD37 did not induce CDC (Figure 2A). The combination of WT
IgG1-CD20-7D8 and WT IgG1-CD37 did not demon- strate enhanced CDC. However, while neither WT IgG1- CD20-11B8 nor WT IgG1-CD37 induced CDC as single agents, the combination promoted strong lysis of approx- imately 60% (Figure 2B). Minimal cell lysis was observed in experiments with heat-inactivated serum, indicating that that cell killing was largely dependent on complement (Online Supplementary Figure S4).
We also examined whether combinations of CD20 and CD37 mAbs with Hx mutations also showed cooperativity in CDC by testing mAb combinations using a full dose-response matrix (8x8 serial dilution grid) based on the EC50 values of the different mAbs. Surprisingly, both Hx-CD20-7D8 and Hx-CD20-11B8 in combination with Hx-CD37 showed enhanced CDC of Daudi cells compared to the single agents (Figure 2C and Online Supplementary Figure S5A). We next assessed whether the observed combination effect was synergistic using the Loewe additivity-based combination index (CI) score cal- culated by CompuSyn, whereby effects were categorized as synergistic (CI<1), additive (CI=1), or antagonistic (CI>1).33 The Loewe additivity-based model assumes syn- ergy when the effect of a drug combination is higher than the effect of a drug combined with itself, and takes into account both the potency and the shape of the dose-effect curve of each drug in the dose-response matrix. Synergy was observed for both Hx-CD20-7D8 and Hx-CD20-11B8 when combined with Hx-CD37, with average CI values of 0.37 and 0.31 (effective dose - ED95), respectively (Figure 2D, Online Supplementary Table S1 and Online Supplementary Figure S5B). At the lower tested mAb con- centrations, synergy was more profound (lower CI values) for combinations of Hx-CD37 with type II CD20 mAb- derived Hx-CD20-11B8 than with type I CD20 mAb- derived Hx-CD20-7D8.
In addition to Daudi cells, we used two other B-cell lines expressing various levels of CD20 and CD37 to further examine the cooperativity in CDC between combinations of Hx-CD37 with Hx-CD20 mAbs or with clinically vali- dated CD20 mAbs. Across all B-cell lines tested, enhanced CDC activity was observed for combinations of Hx-CD37 with Hx-CD20 mAbs, as well as for combinations of Hx- CD37 with ofatumumab, rituximab and obinutuzumab (Figure 2E). Even in WIL2-S cells expressing low levels of CD37, a combination of Hx-CD37 with obinutuzumab induced 72% lysis, whereas the single agents induced only 5% and 10% lysis respectively. Despite high single agent activity of Hx-CD37 and Hx-CD20 mAbs at 10 μg/mL (per mAb) in Daudi cells, the cooperativity between Hx- CD37 and Hx-CD20 mAbs became apparent at the lower mAb concentrations.
Enhanced binding and use of C1q by combinations of hexamerization-enhanced CD20 and CD37 mAbs
We hypothesized that the observed synergy in CDC between Hx-CD20 and Hx-CD37 mAbs resulted from more efficient use of complement proteins, starting with binding of C1q. Therefore, we determined whether com- binations of Hx-CD20 and Hx-CD37 differed in their C1q binding capacity. We incubated Daudi cells with fixed mAb concentrations and titrated C1q, and measured C1q binding and the concentration of C1q required to induce CDC, referred to here as CDC efficacy. Hx-CD20-7D8 already induced efficient C1q binding as a single agent, while Hx-CD37 showed limited C1q binding (Figure 3A).
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