Mechanism of Action of Type II, Glycoengineered, Anti-CD20 Monoclonal Antibody GA101 in B-Chronic Lymphocytic Leukemia Whole Blood Assays in Comparison with Rituximab and Alemtuzumab

Abstract

We analyzed in B-chronic lymphocytic leukemia (B-CLL) whole blood assays the activity of therapeutic mAbs alemtuzumab, rituximab, and type II glycoengineered anti-CD20 mAb GA101. Whole blood samples were treated with Abs, and death of CD19+ B-CLL was measured by flow cytometry. Alemtuzumab efficiently lysed B-CLL targets with maximal lysis at 1–4 h (62%). In contrast, rituximab induced a more limited cell death (21%) that was maximal only at 24 h. GA101 killed B-CLL targets to a similar extent but more rapidly than rituximab, with 19.2 and 23.5% cell death at 4 and 24 h, respectively, compared with 7.9 and 21.4% for rituximab. Lysis by both rituximab and GA101 correlated directly with CD20 expression levels (r2 = 0.88 and 0.85, respectively). Interestingly, lysis by all three Abs at high concentrations was mostly complement dependent, because it was blocked by the anti-C5 Ab eculizumab by 90% in the case of alemtuzumab and rituximab and by 64% in the case of GA101. Although GA101 caused homotypic adhesion, it induced only limited (3%) direct cell death of purified B-CLL cells. Both rituximab and GA101 showed the same efficiency in phagocytosis assays, but phagocytosis was not significant in whole blood due to excess Igs. Finally, GA101 at 1–100 μg/ml induced 2- to 3-fold more efficient NK cell degranulation than rituximab in isolated B-CLL or normal PBMCs. GA101, but not rituximab, also mediated significant NK cell degranulation in whole blood samples. Thus, complement and Ab-dependent cellular cytotoxicity are believed to be the major effector mechanisms of GA101 in whole blood assays.

Lessons for the clinic from rituximab pharmacokinetics and pharmacodynamics

“…36 Indeed, removal of fucose, as is the case with obinutuzumab, results in an increase in its affinity for the FcγRIIIA receptor and increased ADCC. 17,20,37 Conversely, glycosylation is not important for binding to FcRn. Therefore, the Asn297 glycosylation does not affect significantly the half-life of the antibody in vivo.…”

“…Thus, immune cell activation may not be very strong in the circulation where high IgG levels are present. 12,20 Antibodies also interact with the non-specific neonatal FcRn receptor, also known as the Brambell receptor. The binding between IgG and FcRn occurs at the interface between the CH2 and CH3 domains of IgG.…”

“…Obinutuzumab (GA101), a humanized and glycoengineered mAb, shows increased binding to FcγRIIIA and enhanced NK-mediated ADCC, increased direct cell death induction; it is in late-stage clinical trials. 16,17,20 Ofatumumab (HuMax-CD20), a fully human mAb, has increased complement activation potential, particularly in the presence of low CD20 expression levels. 21 Ofatumumab has been tested in clinical trials in CLL patients who are refractory to both fludarabine and alemtuzumab.…”

Regulation of complement and modulation of its activity in monoclonal antibody therapy of cancer

“…Nevertheless, at mAb concentrations, normally reached during mAb therapy, killing of CLL cells in vitro partially depended on complement activation. 52 The anti-CD52 mAb alemtuzumab induced efficient CDC of freshly isolated leukemia/lymphoma cells in vitro 53,54 and complement consumption was demonstrated after infusion of alemtuzumab into patients with B cell lymphoma. 55,56 The anti-CD38 mAb daratumumab was selected for its efficient induction of CDC of multiple myeloma cells 57 and has broad anti-myeloma effects.…”

Lepidopteran cells, an alternative for the production of recombinant antibodies?

“…86 Defucosylation of anti-CD20 or anti-CEA antibodies has been shown however to have no significant effect on phagocytosis, suggesting that receptors other than FcγRIIIA, which are not similarly affected by fucose, play a predominant role in phagocytosis. 102,103 Whereas several antibodies produced in insect cells have been probed for their capacity to activate ADCC, data on phagocytosis by insect cell produced antibodies are even more scarce, with only one report showing activation of monocytes by anticolorectal carcinoma antigen antibody produced in Sf 9 cells. Given the likely importance of this mechanism of action, future work should investigate more systematically the capacity of such antibodies to mediate phagocytosis.…”