Comparison of the in vitro effects of the anti‐CD20 antibodies rituximab and GA101 on chronic lymphocytic leukaemia cells

Abstract

SummaryThe effects of two CD20 antibodies, namely rituximab, the current standard for treatment of chronic lymphocytic leukaemia (CLL) in combination with chemotherapy, and GA101, a glyco-engineered type II antibody were compared on CLL cells ex vivo. Antibody-induced phosphatidylserine exposure was examined in isolated CLL cells. For a more comprehensive assessment of antibody-mediated cell killing including Fc-mediated mechanisms, B cell depletion from whole blood samples was monitored. Treatment with rituximab or GA101 reduced the average viability of isolated CLL cells by 6% or 11%, and the ratio of B to T cells in whole blood samples by 12% or 33%, respectively. Combination with GA101 enhanced the cytotoxicity of the chemotherapeutic agent chlorambucil on isolated CLL cells. CD20 surface expression on CLL cells correlated with GA101-induced B cell depletion, but not with direct cell death induction. Treatment of whole blood samples from CLL patients with a CpG-containing oligonucleotide increased CD20 expression on CLL cells and GA101-dependent B cell depletion. Despite the variable responses of individual CLL samples, the CLL cell depletion from whole blood by GA101 was consistently much stronger than by rituximab, which argues for clinical investigation of GA101 in CLL patients.

Action of novel CD37 antibodies on chronic lymphocytic leukemia cells

“…6,7 Although the marginal DCD induction by rituximab was expected, the average alemtuzumab-induced DCD without crosslinking anti-Fc antibodies and without elevated serum levels may be overestimated in this study owing to three particularly sensitive samples (ID E, J and N). In isolated CLL cells the humanized mAb 37.2 led to similar DCD induction as alemtuzumab, but did not affect T lymphocytes at the same time, thus avoiding a major drawback of alemtuzumab in a clinical setting.…”

“…Subsequently, the samples were examined flow cytometrically for annexin V-binding and 7-amino-actinomycin D (7AAD) staining as previously described. 6 On the average, the percentages of cells showing phosphatidylserine (PS) exposure were significantly increased as compared with untreated samples by all four investigated antibody preparations (Supplementary Figure 1). Annexin V-binding alone was more efficiently induced than PS exposure and membrane disintegration together.…”

“…To prepare these novel CD37 antibodies for a recently started phase I trial of the German CLL study group, we investigated them in comparison with monoclonal antibodies currently used for CLL therapy, that is, rituximab and alemtuzumab, with regard to antibody-induced apoptosis in isolated CLL cells and to B-cell depletion from whole-blood samples as in our recent characterization of the novel CD20 antibody GA101. 6 First, we studied the direct effects of monoclonal antibodies on freshly isolated CLL cells. As immunoreagents targeting CD20 and CD37 were found to trigger programmed cell death in isolated CLL cells in a caspase-independent manner, 3,7 we wish to refer to this type of antibody-induced cell killing as DCD, so as to distinguish it from classical, caspase-dependent, for example, DNA damage-induced apoptosis.…”

“…Annexin V-binding alone was more efficiently induced than PS exposure and membrane disintegration together. Relative viability and DCD induction values were derived from these primary data as described, 6 and are detailed for individual CLL samples (Supplementary Table 1). The mean DCD induction according to PS exposure in the complete set of samples was highest by mAb 37.1, with 51% followed by alemtuzumab and mAb 37.2 with 35 and 32%, respectively, and by far exceeding that by rituximab.…”

Glycoengineering of Therapeutic Antibodies Enhances Monocyte/Macrophage-Mediated Phagocytosis and Cytotoxicity

“…However, the different experimental conditions used in the two studies (30 min ADCP, E:T ratio of 1:5 in Rafiq et al versus 4 h ADCP, E:T ratio of 3:1 in the current study) and differences in phagocytic effector cells (monocytes and MDMs derived from patients with CLL in Rafiq et al versus CD16 + /CD16 2 monocytes and M1/M2c macrophages in the current study) might have led to different results. Importantly, the elimination of leukemic B cells in whole blood from patients with CLL is consistently superior with GA101 (41,42).…”