For over a decade, anti-CD20 monoclonal antibodies (mAbs) have successfully been used in the therapy of B-cell malignancies. Obinutuzumab (GA101), a type-II glycoengineered anti-CD20 mAb approved for treatment of Chronic Lymphocytic Leukaemia (CLL) and Follicular Lymphoma, was designed to have enhanced antibody-dependant cellular cytotoxicity compared with Rituximab. Pre-clinical data showed that in addition, GA101 also exhibited significantly enhanced direct cell death. The complete mechanism of increased direct cell death is still unknown.

The biological role of CD20 remains unclear but it has been shown to act as part of an ion channel complex, a component of the store operated calcium (Ca2+) entry system. Numerous studies have investigated the immediate impact of anti-CD20 mAbs on intracellular Ca2+. Yet to date, none have assessed intracellular Ca2+ over a longer period.

To investigate the effect of anti-CD20 mAbs on intracellular Ca2+, we initially used 4 B-cell lymphoma cell lines to assess Ca2+ flux immediately after treatment with either Rituximab or GA101. Cells were first stained with a fluorescent calcium indicator and baseline measurements recorded by flow cytometry. Cells were then treated with each antibody and fluorescence measurements were recorded for a further 5-10 minutes. Next, we conducted a time course experiment to visualise the kinetics of intracellular Ca2+ after 1, 2, 4, 8, and 24 hour treatments with either Rituximab or GA101; Trastuzumab was used as an isotype control. After treatment, samples were stained with a labelled calcium indicator before quantifying intracellular Ca2+ using imaging flow cytometry. We validated our findings in primary CLL samples using the same method. We conducted liquid chromatography tandem mass spectrometry based label-free phosphoproteomics on SUDHL4 cells in biological triplicates after 1 and 24 hour treatments with GA101 and Rituximab. Expression levels of >8500 phosphorylation sites were measured across treatments to establish changes in intracellular signalling and kinase activity.

In line with published data, we observed an influx of Ca2+ when cells were treated with Rituximab but intracellular Ca2+ was not significantly altered for any of the extended treatments. In contrast, GA101 treatment did not induce an immediate influx of Ca2+,and a reduction in intracellular Ca2+ was observed after 1 hour treatment which was sustained across all extended treatment periods. This difference in kinetics was validated in primary CLL samples. Global phosphoproteomic analysis found that the activity of Polo-Like Kinase 2 (PLK2) was significantly increased following treatment with GA101. The increase in the activity of this kinase was specific to GA101 and was unchanged following treatment with Rituximab. Simultaneously there was a significant increase in the phosphorylation of Calumenin at Ser44, a known binding site of PLK2. Samples treated with GA101 for 24 hours exhibited a larger downregulation of pathways associated with calcium signalling compared to Rituximab-treated and control samples.

Calumenin, a member of the CREC family, is able to bind 7 Ca2+ ions. It can be localised in the cytosol, in compartments of the secretory pathway, on the plasma membrane or in the extracellular space. We hypothesise that once phosphorylated, this protein bound with Ca2+ is secreted extracellularly, resulting in reduced intracellular Ca2+.

In summary we report for the first time that intracellular Ca2+ is significantly decreased as a result of GA101 treatment in cell lines and primary samples. Using a global unbiased approach we have identified a significant increase in PLK2 activity and we observed a large increase in phosphorylation of Calumenin. Our data suggests that these proteins may contribute to the increased direct cell death observed as a result of GA101 treatment. We believe that expression and activity of these proteins are crucial in order to maximise GA101-mediated direct cell death. Ongoing studies are examining the functional roles of PLK2 and Calumenin in the reduction of intracellular Ca2+, comparing Ca2+ levels in response to antibody treatment in primary samples from different CLL subtypes, and assessing and comparing levels of apoptosis following antibody treatment to see whether there is a correlation with reduced intracellular Ca2+.


Gribben: Celgene: Honoraria; TG Therapeutics: Honoraria; Genentech/Roche: Honoraria; Karyopharm: Honoraria; Kite: Honoraria; Abbvie: Honoraria; Pharmacyclics: Honoraria; Janssen: Honoraria; Acerta: Honoraria.

Author notes


Asterisk with author names denotes non-ASH members.