Ab binding to CD20 has been shown to induce apoptosis in B cells. In this study, we demonstrate that rituximab sensitizes lymphoma B cells to Fas-induced apoptosis in a caspase-8-dependent manner. To elucidate the mechanism by which Rituximab affects Fas-mediated cell death, we investigated rituximab-induced signaling and apoptosis pathways. Rituximab-induced apoptosis involved the death receptor pathway and proceeded in a caspase-8-dependent manner. Ectopic overexpression of FLIP (the physiological inhibitor of the death receptor pathway) or application of zIETD-fmk (specific inhibitor of caspase-8, the initiator-caspase of the death receptor pathway) both specifically reduced rituximab-induced apoptosis in Ramos B cells. Blocking the death receptor ligands Fas ligand or TRAIL, using neutralizing Abs, did not inhibit apoptosis, implying that a direct death receptor/ligand interaction is not involved in CD20-mediated cell death. Instead, we hypothesized that rituximab-induced apoptosis involves membrane clustering of Fas molecules that leads to formation of the death-inducing signaling complex (DISC) and downstream activation of the death receptor pathway. Indeed, Fas coimmune precipitation experiments showed that, upon CD20-cross-linking, Fas-associated death domain protein (FADD) and caspase-8 were recruited into the DISC. Additionally, rituximab induced CD20 and Fas translocation to raft-like domains on the cell surface. Further analysis revealed that, upon stimulation with rituximab, Fas, caspase-8, and FADD were found in sucrose-gradient raft fractions together with CD20. In conclusion, in this study, we present evidence for the involvement of the death receptor pathway in rituximab-induced apoptosis of Ramos B cells with concomitant sensitization of these cells to Fas-mediated apoptosis via Fas multimerization and recruitment of caspase-8 and FADD to the DISC.

IntroductionDespite advances in T-cell leukemia therapy, only a minority of patients achieves long term tumor-free survival with conventional chemotherapy at the cost of significant and often irreversible toxic side effects. 1 Therefore, new therapeutic approaches with enhanced tumor selectivity and more favorable toxicity profiles are urgently needed. Several promising targeted approaches have been developed, including naked antibodies, 2,3 immunotoxins, 4,5 and various cancer-selective small inhibitory molecules. 6,7 Furthermore, certain members of the tumor necrosis factor (TNF) superfamily show promising proapoptotic activity toward various human leukemias and lymphomas.Fas ligand (FasL), a prominent member of the TNF superfamily, shows superior antileukemia activity. FasL is present on lymphocytes and monocytes/macrophages as a type II transmembrane protein, (memFasL). Fas, the cognate receptor for FasL, belongs to the family of transmembrane proteins known as death receptors. Death receptors can detect the presence of specific extracellular death signals and rapidly trigger cellular destruction by apoptosis. Fas expression at the cell surface is observed in biopsies and cell lines derived from a variety of tumors. Moreover, the antitumoral effects of various chemotherapeutic drugs have been attributed partly to p53-mediated up-regulation of Fas signaling is also known to be a key element in the effector phase of a cytotoxic T lymphocyte (CTL) response against tumor cells.Like other members of the TNF superfamily, the extracellular domain of FasL can be proteolytically cleaved into a soluble homotrimeric form [13][14][15].Early attempts to exploit Fas agonists such as anti-Fas antibodies and multimeric recombinant FasL preparations for therapy revealed extremely potent tumoricidal effects toward isolated primary tumor cells and cell lines. 13,[16][17][18][19] However, in vivo application of most Fas antagonists was associated with acute lethality in mice, 20-22 thereby excluding therapeutic evaluation in humans. Nevertheless, the principal feasibility of therapeutic Fas activation in cancer therapy was clearly demonstrated in mice that lack a functional FasL/Fas system (lpr/gld mice), 23 and by treatment of xenografted tumors with human Fas-specific antibodies. 24 Recent studies have revealed that certain recombinant FasL preparations contain oligomeric, multimeric, and even aggregated sFasL forms and that these forms are responsible for the observed systemic toxicity. 25 In contrast, homotrimeric sFasL is not toxic to normal cells and may even antagonize the function of membranebound FasL. [25][26][27] Importantly, homotrimeric sFasL also lacks tumoricidal activity. However, selective delivery of sFasL to a predetermined tumor-associated target antigen can restore the full apoptotic potential of sFasL. 28 Recently, we demonstrated that the leukemia selectivity of homotrimeric TRAIL (TNF-related apoptosis-inducing ligand), Supported by grants from the Dutch Cancer Society (RUG 2002(RUG -2668(RUG and 20...

Although the treatment outcome of lymphoid malignancies has improved in recent years by the introduction of transplantation and antibody-based therapeutics, relapse remains a major problem. Therefore, new therapeutic options are urgently needed. One promising approach is the selective activation of apoptosis in tumor cells by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). This study investigated the pro-apoptotic potential of a novel TRAIL fusion protein designated scFvCD19:sTRAIL, consisting of a CD19-specific single-chain Fv antibody fragment (scFv) fused to the soluble extracellular domain of TRAIL (sTRAIL). Potent apoptosis was induced by scFvCD19:sTRAIL in several CD19-positive tumor cell lines, whereas normal blood cells remained unaffected. In mixed culture experiments, selective binding of scFvCD19:sTRAIL to CD19-positive cells resulted in strong induction of apoptosis in CD19-negative bystander tumor cells. Simultaneous treatment of CD19-positive cell lines with scFvCD19:sTRAIL and valproic acid (VPA) or Cyclosporin A induced strongly synergistic apoptosis. Treatment of patient-derived acute B-lymphoblastic leukemia (B-ALL) and chronic B-lymphocytic leukemia (B-CLL) cells resulted in strong tumoricidal activity that was further enhanced by combination with VPA. In addition, scFvCD19:sTRAIL prevented engraftment of human Nalm-6 cells in xenotransplanted NOD/Scid mice. The pre-clinical data presented here warrant further investigation of scFvCD19:sTRAIL as a potential new therapeutic agent for CD19-positive B-lineage malignancies.

The clinical efficacy of the CD20-specific chimeric monoclonal antibody rituximab is significantly hampered by intrinsic or acquired resistance to therapy. Rituximab activates antibodydependent cellular cytotoxicity/complement-dependent cytotoxicity-dependent lysis but also induces apoptosis by cross-linking of its target antigen CD20. Recent reports indicate that this apoptotic activity of rituximab can be synergized by cotreatment with Fas agonists. Here, we report on a strategy designed to exploit and optimize the synergy between rituximab and Fas signaling by genetically fusing a rituximab-derived antibody fragment to soluble Fas ligand (sFasL). The resultant fusion protein, designated scFvRit:sFasL, potently induced CD20-restricted apoptosis in a panel of malignant B-cell lines (10 of 11) and primary patient-derived malignant B cells (two of two non-Hodgkin lymphoma and five of six B cell chronic lymphocytic leukemia). ScFvRit:sFasL efficiently activated CD20 and Fas apoptotic signaling, resulting in a far superior proapoptotic activity compared with cotreatment with rituximab and Fas agonists. ScFvRit:sFasL lacked activity toward normal human B cells and also lacked systemic toxicity in nude mice with no elevation of aspartate aminotransferase and alanine aminotransferase levels or liver caspase-3 activity. In conclusion, scFvRit:sFasL efficiently activates CD20 and Fas-apoptotic signaling and may be useful for the elimination of malignant B cells. [Cancer Res 2008;68(2):597-604]