Ofatumumab: Evaluation of in-vitro response mechanisms

Introduction: The application of monoclonal antibodies has become standard of care in the treatment of Chronic lymphocytic leukemia (CLL). We compared the single-agent-activity of three currently approved therapeutic antibodies for CLL, namely Rituxi-mab, Alemtuzumab, and Ofatumumab. Question: Our goal was to identify underlying mechanisms of action of monoclonal antibodies in CLL in order to recognize potential limitations of their use and to develop strategies to overcome these limitations or at least to use the antibodies in the most beneficial way. We had our major focus on the impact of genetic subgroups of CLL and on Ofatumumab, a relatively new CD20-antibody in comparison with Rituximab and Alemtuzumab. Method: Patient samples were analysed by flow cytometry, fluorescence in-situ hybridiziation, liquid chromatography, and classic sequencing. Antibody-treatment was performed in both cell culture and whole blood assays. We assessed CLL-cell-viability, homotypic adhesion, potential synergism with other substances, and the impact of the cellular micro-environment by co-culturing CLL cells with fibroblastoid stroma cells. In parallel, CLL-cell-concentration, and effector consumption were assessed. Read out methods comprised microscopy, multi-color flow cytometry and luminometry. Results: Rituximab, Alemtuzumab, and Ofatumumab showed varying activity and different mechanisms of action. Regarding each antibody individually, no considerable difference could be detected among different risk-stratified CLL subgroups. In a sub-group of patients, we mimicked in vivo micro-environment by coincubation of CLL cells with HS5-cells and discovered a decrease of Alemtuzumab-activity but not Ofatumumab-activity. Summary: Ofatumumab seems highly suitable for various combinations and high-risk CLL patients might benefit of those. Based on our in vitro findings, we suggest several strategies to enhance in vivo Ofatumumab activity