Abstract
Multiple myeloma is still an incurable plasma malignancy because patients are prone to quickly relapse after conventional treatment or even high-dose chemotherapy. At the present time, monoclonal antibodies (mAbs) are being successfully used to treat cancers. We recently discovered that anti-β2-microglobulin (β2M) mAbs (IgG1 isotype) induced myeloma cell apoptosis (
Yang et al., Cancer Cell 2006; 10:295–307
). The mAbs bind, cross-link, and recruit surface β2M/MHC class I molecules on myeloma cells to lipid rafts where downstream signaling pathways are activated. Therefore, it is possible that enhancing the capacity of the mAbs to cross-link surface MHC class I could further improve the efficacy of mAb-induced myeloma cell apoptosis. To examine this hypothesis, we generated anti-β2M mAbs of IgM isotype with a pentameric structure. By using Annexin V and TUNEL assays, we showed that, compared with monomeric IgG mAbs, IgM anti-β2M mAbs exhibit stronger tumoricidal activity on all six myeloma cell lines and primary myeloma cells isolated from five myeloma patients in dose- and time-dependent manner. About 80% to 90% of myeloma cells were apoptotic when treated with IgM mAb at a concentration as low as 20 mg/mL in a 12-hour culture. Furthermore, IgM anti-β2M mAbs, which at the same dose had stronger therapeutic efficacy than IgG mAbs in vivo, had greatly reduced tumor burdens and retarded tumor growth in SCID mice. To examine whether the pentameric structure plays an important role in IgM mAb-mediated tumoricidal activity, IgM mAbs were treated with β-mercaptoethanol (2ME), a specific agent that breaks the J-chain of IgM antibody leading to irreversible disruption of the IgM pentameric structure. By using native gel electrophoresis, we confirmed that without 2ME treatment, IgM mAbs were pentamers with one band of 950 kDa. After treatment with 2ME, IgM mAbs displayed a strong band in 175 kDa (monomer), indicating that 2ME completely broke pentamers to monomers. Furthermore, we found that the addition of 2ME-pretreated IgM mAbs to cell culture induced much weaker myeloma cell apoptosis than pentameric IgM mAbs. By using Western blot analysis, we further showed that 2ME-pretreated IgM mAbs induce weaker JNK activation and caspase-9, -8, -3, and PARP cleavage. By using confocal microscopy, we showed that 2ME-pretreated IgM mAbs are much less efficient at recruiting β2M/MHC class I molecules into lipid rafts on myeloma cells, although 2ME-pretreated IgM mAbs bound well to myeloma cell surface. Thus, our findings indicate that enhancing cross-linking of surface β2M/MHC class I molecules may be a novel approach to improve the antimyeloma efficacy of the mAbs. This study also suggests that our IgM anti-β2M mAbs may be a better therapeutic agent for future clinical application.Disclosures: No relevant conflicts of interest to declare.
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2008, The American Society of Hematology
2008
