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...

mAbs are becoming increasingly utilized in the treatment of lymphoid disorders. Although Fc-FcγR interactions are thought to account for much of their therapeutic effect, this does not explain why certain mAb specificities are more potent than others. An additional effector mechanism underlying the action of some mAbs is the direct induction of cell death. Previously, we demonstrated that certain CD20-specific mAbs (which we termed type II mAbs) evoke a nonapoptotic mode of cell death that appears to be linked with the induction of homotypic adhesion. Here, we reveal that peripheral relocalization of actin is critical for the adhesion and cell death induced by both the type II CD20-specific mAb tositumomab and an HLA-DRspecific mAb in both human lymphoma cell lines and primary chronic lymphocytic leukemia cells. The cell death elicited was rapid, nonapoptotic, nonautophagic, and dependent on the integrity of plasma membrane cholesterol and activation of the V-type ATPase. This cytoplasmic cell death involved lysosomes, which swelled and then dispersed their contents, including cathepsin B, into the cytoplasm and surrounding environment. The resulting loss of plasma membrane integrity occurred independently of caspases and was not controlled by Bcl-2. These experiments provide what we believe to be new insights into the mechanisms by which 2 clinically relevant mAbs elicit cell death and show that this homotypic adhesion-related cell death occurs through a lysosome-dependent pathway.Introduction mAbs are becoming increasingly utilized in the treatment of lymphoid disorders (1, 2). In particular, mAb directed to cell-surface antigens on malignant B cells has proven the most clinically effective, with the anti-CD20 mAb, rituximab, being the first to be approved by the US FDA for the treatment of cancer. Rituximab has substantially improved outcome for patients with many different types of non-Hodgkin lymphoma and has now been administered to over 1 million patients in the decade since its approval. Despite such success, treatment is not curative and there is intense preclinical and clinical investigation of many other engineered mAbs directed to both CD20 and a host of other cell-surface antigens (2).

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p53 mutant tumour cells respond to genotoxic insults by bypassing G1 arrest and halting in G2. Following release from G2 arrest they undergo mitotic catastrophe, whereby mitotic cycling is suppressed, delayed apoptosis begins and endopolyploid cells are produced. The ability of these endopolyploid cells to participate in the restitution process is controversial. To facilitate recovery, these endopolyploid cells must repair the extensive DNA damage induced. DNA damage and its resolution were studied by observing the kinetics of γ-H2AX foci formation and by comet assay analysis. Subsequently, the kinetics and distribution of Rad51 foci were studied as a measure of homologous recombination. Here we present evidence of the resolution of DNA damage in endopolyploid cells through a decrease of tail moment by comet assay and in the number of cells expressing γ-H2AX foci. Rad51 foci expression reached a maximum in endopolyploid cells on days 5-6 after irradiation, when delayed apoptosis was maximal, indicating that cells were being selected for survival at this time. Furthermore, the proportion of Annexin-V-positive polyploid cells decreased as they continued ongoing rounds of DNA replication, suggesting endoreduplication is involved in selecting cells resistant to apoptosis. Our findings suggest that after severe genotoxic insult endopolyploid cells have a transient survival advantage that may contribute to radioresistance of tumours that undergo mitotic catastrophe.