Combination Therapy with FTY720 (Fingolimod) and Bispecific Anti-CD20/CD74 Antibodies in Mantle Cell Lymphoma,

Mantle cell lymphoma (MCL) is one of the most challenging B-cell lymphomas to treat. Although the response rates with first-line conventional or high-dose chemotherapy, with or without stem-cell transplantation, are high, most patients relapse, after which their prognosis is considered poor. Thus, new therapeutic interventions in MCL are needed, among which targeted therapy with a variety of monoclonal antibodies (mAbs), either alone or in combination with other biological agents or drugs, is a major focus of ongoing clinical studies. FTY720 (fingolimod), an immunosuppressive agent approved by the FDA as a frontline treatment for relapsing multiple sclerosis, shows promising pre-clinical activity in chronic lymphocytic leukemia and MCL. More recently, FTY720 was also found to increase CD74 expression and sensitize MCL cells to milatuzumab (anti-CD74 humanized antibody)-mediated cell death (Alinari L, et al. ASH 2011 Abstract submitted). Here we report that the in vitro cytotoxicity of two novel bispecific anti-CD20-/CD74 antibodies to MCL lines can be enhanced significantly by combining with FTY720. Methods: The two bispecific, anti-CD20/CD74, hexavalent antibodies (HexAbs), designated 20-(74)-(74) and 74-(20)-(20), were generated from veltuzumab (anti-CD20 humanized mAb) and milatuzumab with the Dock-and-Lock (DNL) method. The effect of FTY720 on cell-surface expression of CD20, CD22, CD74, and HLA-DR was assessed by flow cytometry after treating JeKo-1, Granta-519 and Mino MCL cell lines with FTY720 for 18 h followed by labeling the cells with primary antibody and FITC-conjugated secondary antibody at 4°C. The in vitro cytotoxicity of the combination treatment was determined by Annexin V /PI binding assays and the data analyzed by the Chou-Talalay equation to calculate the combination index (CI). Results: In the three MCL lines tested, FTY720 at 12.5 μM induced almost 9-fold increase of CD74, a modest decrease of CD20, and no apparent change in CD22 and HLA-DR, when compared to untreated controls. Further studies in JeKo-1 incubated with varying doses of FTY720 revealed a 5-fold enhanced expression of CD74 could be achieved at 4 μM, but no notable changes were observed with FTY720 at 2.5 μM, or below. The viability of JeKo-1 cells treated with a single agent for 16 h was determined by the Annexin V/PI staining assay to be 45% for FTY720 at 4 μM and 65–70 % for either 20-(74)-(74) or 74-(20)-(20) at 33 nM. Combined treatments with FTY720 and 20-(74)-(74) at 4 μM and 33 nM, respectively, resulted in about 15% live cells (Annexin V-negative/PI-negative) with statistically significant P values (<0.01) compared to 20-(74)-(74) and FTY720 alone. For FTY720 and 74-(20)-(20), the combination treatment resulted in 21% live cells, also with statistically significant P values (<0.01) compared to each agent alone. Initial analysis of these data yielded a CI of <0.87, suggesting a synergistic effect with the combination therapy. Conclusion: The anti-MCL activity of 20-(74)-(74) and 74-(20)-(20) can be further enhanced by FTY720, as demonstrated for JeKo-1. Studies are underway to evaluate similar cytotoxic effects in xenograft mouse models of MCL and to define the signaling pathways triggered by combining FTY720 with the bispecific anti-CD20/CD74 HexAbs.

Gupta:Immunomedics, Inc.: Employment. Michel:Immunomedics, Inc.: Employment. Goldenberg:Immunomedics, Inc.: Consultancy, Employment, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Chang:Immunomedcis, Inc.: Employment, Patents & Royalties, Stock Options.