No abstract

The anti-CD20 mAb rituximab is central to the treatment of B-cell malignancies, but resistance remains a significant problem. We recently reported that resistance could be explained, in part, by internalization of rituximab (type I anti-CD20) from the surface of certain B-cell malignancies, thus limiting engagement of natural effectors and increasing mAb consumption. Internalization of rituximab was most evident in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), but the extent of internalization was heteroge- IntroductionThe anti-CD20 mAb rituximab has improved the overall survival of patients with follicular (FL) and diffuse large B-cell lymphoma (DLBCL). [1][2][3][4] However, in MCL, only modest responses are seen 5 and in CLL, fludarabine, cyclophosphamide and rituximab (FCR) therapy delivers improved responses but has yet to show a similar improvement in overall survival, 6 albeit the current follow-up is relatively short. Interestingly, those responses seen in CLL have often been achieved with high doses of rituximab, 6 suggesting that more mAb is needed to coat the targets or that it is consumed in some way. Even within rituximab-responsive lymphomas, a proportion of cases show resistance on first treatment with rituximab or eventually become resistant to rituximab-containing combination therapy (reviewed in Stolz et al 7 ). The molecular basis of this resistance and the observed sensitivity of different lymphoma subtypes is unclear (reviewed in Lim et al 8 ), but is highly relevant to improving outcomes.In addition to understanding target resistance, many groups are working to deliver anti-CD20 mAb reagents with improved affinity and more potent engagement of cytotoxic effectors. Anti-CD20 mAb can be defined as type I (eg, rituximab, ofatumumab) or type II (eg tositumumab, GA101), according to their ability to redistribute CD20 into lipid rafts in the plasma membrane and function in various effector assays. 9-11 It is still not clear what characteristics are required for the optimal reagent, but it is generally accepted that Fc:Fc ␥ receptor (Fc␥R) interactions are crucial to the efficacy of anti-CD20 mAb. [12][13][14][15] In particular, Fc␥RIIIa on myeloid effectors appears critical in controlling Ab potency and in keeping with this, lymphoma patients bearing the higher affinity 158V allele in Fc␥RIIIa respond better to rituximab compared with those with the low affinity 158F allotype, 16 leading many investigators to focus on augmenting the interaction of mAb with Fc␥RIIIa, for example via defucosylation. 17 Less attention has been given to the potential effects of the ITIM-containing inhibitory Fc␥R, Fc␥RIIb. Fc␥RIIb is a negative regulator of ITAM-containing receptors, such as the B-cell receptor (BCR) and the activatory Fc␥R. 18 Most hematopoietic cells coexpress inhibitory and activatory Fc␥R, and tumors are reported to be more sensitive to mAb immunotherapy in Fc␥RII Ϫ/Ϫ mice because of the removal of the inhibitory restraint of this receptor from myeloid effectors such as macro...

The anti-CD20 mAb rituximab has substantially improved the clinical outcome of patients with a wide range of B-cell malignancies. However, many patients relapse or fail to respond to rituximab, and thus there is intense investigation into the development of novel anti-CD20 mAbs with improved therapeutic efficacy. Although Fc-Fc␥R interactions appear to underlie much of the therapeutic success with rituximab, certain type II anti-CD20 mAbs efficiently induce programmed cell death (PCD), whereas rituximab-like type I anti-CD20 mAbs do not. Here, we show that the humanized, glycoengineered anti-CD20 mAb GA101 and derivatives harboring non-glycoengineered Fc regions are type II mAb that trigger nonapoptotic PCD in a range of B-lymphoma cell lines and primary B-cell malignancies. We demonstrate that GA101-induced cell death is dependent on actin reorganization, can be abrogated by inhibitors of actin polymerization, and is independent of BCL-2 overexpression and caspase activation. GA101-induced PCD is executed by lysosomes which disperse their contents into the cytoplasm and surrounding environment. Taken together, these findings reveal that GA101 is able to potently elicit actin-dependent, lysosomal cell death, which may potentially lead to improved clearance of B-cell malignancies in vivo. IntroductionThe addition of the anti-CD20 mAb rituximab to chemotherapy has substantially improved the clinical outcome for many patients with a wide range of B-cell malignancies. 1-3 However, despite the unprecedented success of rituximab, a substantial proportion of patients with CD20-positive B-cell malignancies fail to achieve a complete remission or relapse after receiving rituximab-containing immunochemotherapy. 4 These areas of unmet clinical need highlight the requirement to develop improved treatments for these patients. Given both the success with rituximab and the rapid development of mAb engineering technology, there is currently intense investigation into the development of novel anti-CD20 mAbs aimed at improving therapeutic efficacy. Central to this challenge, is an enhanced understanding of the mechanism of action of anti-CD20 mAbs.Anti-CD20 mAbs can activate a range of potential tumor cell killing pathways (reviewed in Lim et al 5 ) including Fc-Fc␥ receptor (Fc␥R) interactions (namely Ab-dependent cellular cytotoxicity [ADCC] and phagocytosis mediated by Fc␥R-expressing immune effector cells such as macrophages and/or NK cells), complement-dependent cytotoxicity (CDC), or the direct induction of programmed cell death (PCD). Although it is well established that Fc-Fc␥R interactions are critical for the in vivo efficacy of anti-CD20 mAbs, 6-8 the role of complement remains disputed as to whether it is beneficial, 9,10 inconsequential, 7,11,12 or even detrimental to anti-CD20 mAb efficacy. 13,14 However, the potential importance of PCD in enhancing anti-CD20 mAb potency remains largely underinvestigated, perhaps because it does not appear to play a major role in the therapeutic efficacy of rituximab. 15 We have c...