Christian Kodele, Lian Li, Jane Yang, University of Utah
Non-Hodgkin lymphoma (NHL) is an immune disease mostly of B-cell origin (eighty-five percent of the time) as well as the ninth leading cause of cancer death in the United States. Although treatments for NHLs greatly improved following the FDA approval of Rituximab (RTX), refractive malignancies still occur that are nonresponsive and/or resistance to current therapies in at least a third of all patients. This has been attributed both to the inability of immune effector cells (eg., macrophages, natural killer cells) to hypercrosslink ligated monoclonal antibodies (mAbs), and to Fc receptor (FcR)-mediated endocytosis or ÛÏtrogocytosisÛ of CD20 antigens. In order to address these clinical obstacles, we designed a novel paradigm in macromolecular therapeutics that can specifically kill cancer cells without a drug. This paradigm is based on the use of anti-CD20 Fab’ fragments in a multivalent system. Crosslinking of CD20 receptors leads to receptor clustering, transfer to lipid rafts, opening of a calcium channel, and ultimately apoptosis. Additionally, the removal of the Fc fragment resulted enticingly in both the rendering of the system to be immune dependent and in decreasing the numerous adverse effects. In this study, we have used human serum albumin (HSA) as the multivalent carrier of RTX based Fab’ fragments. We have covalently attached multiple Fab’ fragments to HSA, characterized the nanoconjugate’s physiochemical properties, and evaluated its efficacy to induce apoptosis of Raji B cells in vitro. The efficacy of the nanoconjugate to induce apoptosis was determined with Annexin V assay and flow cytometry. The interaction of the nanoconstruct with Raji cells was characterized using confocal microscopy of Cy5 labeled conjugates. As predicted, the HSA-(Fab’)x conjugate was able to induce cell death in vitro. The results of the Annexin V apoptosis assay showed that 38.9 percent of the cell population treated with the conjugate became apoptotic, while 13.6 and 15.7 percent of the cell populations untreated and treated with whole RTX mAb became apoptotic respectively. Furthermore, images recorded by use of confocal microscopy suggest that the attachment of HSA-(Fab’)x conjugate to the cell membrane is CD20 specific. While not conclusive, the combination of these results suggest that the mechanism of action involves cross-linking of the CD20 receptor, which subsequently induces apoptosis. We believe these results warrant further investigation of the mechanism of action of HSA-(Fab’)x, as well as the treatment potential of this nanoconjugate.