Superior Activity of Fusion Protein scFvRit:sFasL over Cotreatment with Rituximab and Fas Agonists

Abstract

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]

Two Structurally Different Rituximab-Specific CD20 Mimotope Peptides Reveal That Rituximab Recognizes Two Different CD20-Associated Epitopes

“…Nonetheless, the inhibition of rituximab binding by Rp5-L (and Rp15-C) was also observed with other human B lymphoid cells (Ramos and LG-2) and with primary CD20 ϩ lymphocytes (data not shown). Moreover, an inhibitory effect in the human Burkitt's lymphoma cell line BJAB was recently reported for the Rp5-Lfamily peptide Rp10-L (bearing the Rp5-L motif ͳWPxWLEʹ (13, 16)), which competed with the binding of a rituximab-derived construct (36).…”

The Role of Complement in the Mechanism of Action of Rituximab for B-Cell Lymphoma: Implications for Therapy

“…A genetically engineered fusion protein, scFvRit:sFasL, containing a rituximab-derived antibody fragment and Fas ligand was successfully used in malignant B cells [129] (Fig. 4).…”

The Role of FasL and Fas in Health and Disease

“…In accordance with this hypothesis, we and others showed that fusion proteins composed of an N-terminal cell surface antigen-recognizing single chain fragment (scFv) and soluble FasL exert on antigen positive cells a several order of magnitude higher activity than on antigen negative cells. [265][266][267] Thus, cell surface retention of scFv-FasL fusion proteins by interaction with their cognate cell surface antigens constitutes pseudo-membrane FasL (see Fig. 5).…”