Multivalent Human Serum Albumin - Rituximab Fab' Conjugates Induce Apoptosis in Lymphoma Cells

honors thesisCollege of EngineeringBiomedical EngineeringJindřich KopečekNon-Hodgkin lymphoma (NHL) is an immune disease, mostly of B-cell origin (85%), 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 resistant 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 "trogocytosis" 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 low molecular weight 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, opening of a calcium channel, deactivation of mitochondrial activity, and ultimately apoptosis. Additionally, the removal of the Fc fragment has the enticing potential to both render the system to be immune independent as well as decrease 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, calcium influx, and mitochondrial potential reduction of Raji B cells in-vitro. The interaction of the nanoconstruct with Raji cells was characterized using confocal microscopy of Cy5 labeled conjugates. The efficacy of the nanoconjugate to induce the aforementioned events was determined with Annexin V/PI assay, Fluo-3 AM assay, and JC-1 assay respectively, followed by flow cytometry. As predicted, the HSA-(Fab')x conjugate was able to induce cell death in-vitro. The results of the Annexin V/PI apoptosis assay showed that 38.9% of the cell population treated with the conjugate became apoptotic, while 13.6% and 15.7% of the cell populations untreated and treated with whole RTX mAb became apoptotic, respectively. The conjugate also proved to induce calcium influx as well as reduce mitochondrial potential in Raji cells. The results of these assays showed a disparate fluorescence intensity (Fluo-3 AM) as well as fluorescence ratio (JC-1) between the conjugate and the adjacent controls, albeit not statistically significant in the JC-1 examination. Furthermore, images recorded by use of confocal microscopy suggest that the binding 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