Ofatumumab, a Fully Human Monoclonal Antibody Targeting CD20, Demonstrates Activity Against and Potentiates the Anti-Tumor Activity of Chemotherapy Agents In Rituximab-Sensitive Cell Lines (RSCL), Rituximab-Resistant Cell Lines (RRCL), Lymphoma Xenografts, and Primary Tumor Cells Derived From Patients with B-Cell Non-Hodgkin Lymphoma (NHL)

Abstract 3917

Treatment with the anti-CD20 monoclonal antibody rituximab (RTX) has become the standard-of-care for the treatment of B-cell non-Hodgkin lymphoma (NHL). Despite revolutionizing NHL therapy, many patients demonstrate resistance de novo or develop resistance to RTX following treatment with RTX-containing regimens and/or RTX-based maintenance schedules. Ofatumumab (OFA) is a new 2^nd-generation CD20 mAb targeting a novel membrane-proximal epitope on the CD20 antigen. OFA has been FDA-approved for the treatment of fludarabine- and alemtuzumab-refractory CLL and is being evaluated in several clinical trials in NHL. To better define OFA's activity, we conducted pre-clinical studies comparing OFA vs. RTX in a panel of RSCL, RRCL, primary lymphoma cells (n=10), and in a lymphoma xenograft model. Antibody-dependant cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) assays were performed to evaluate differences in activity between RTX and OFA. Lymphoma cells were labeled with ⁵¹Cr prior to incubation with RTX or OFA (1 or 10 mg/ml) plus effector cells or human serum respectively. ⁵¹Cr-release was measured and the percentage of lysis was calculated. In addition, we evaluated the effect of OFA in the cytotoxic effects of chemotherapy agents (doxorubicin, cisplatin and vincristine) and correlated OFA anti-tumor activity to biomarkers known to affect RTX activity (i.e. CD20, CD55, and CD59 surface expression) using qualitative and quantitative flow cytometry. Competitive binding assays were performed using fluorescent-labeled RTX or OFA. OFA was more potent than RTX in elucidating effective CDC at the doses tested not only in RSCL, but also in all RRCL and in primary tumor cells derived from patients with B-cell lymphoma. OFA and RTX were equally effective in ADCC assays. In RSCL and RRCL, there was a linear decrease in sensitivity to RTX (as evaluated by CDC) with decreasing CD20 expression; in contrast, OFA maintained activity even at the lowest levels of CD20 expression. Furthermore, OFA was active despite high levels of CD59 and CD55. OFA had a higher affinity for CD20 than RTX in RSCL. Pre-incubation of RSCL and RRCL with OFA enhanced the anti-tumor activity of chemotherapy agents as determined by alamar blue reduction. Severe combined immunodeficiency (SCID) mice were inoculated via tail vein with Raji cells (day 0) and assigned to observation versus 4 doses of either OFA or RTX (1 or 10mg/kg/dose). The end point of the study was overall survival. Statistical analysis was performed with Kaplan-Meier survival curves and P values calculated by log-rank test. OFA was more effective in controlling in vivo lymphoma growth than RTX. The median survival for animals treated with OFA (1 or 10mg/kg/dose) [73 days and 78 days] was longer than those treated with RTX [56 days and 61 days] (P=0.04 and P=0.04 respectively). Our data suggest that OFA is more potent than RTX not only in RTX-sensitive but also in RTX-resistant models and potentiates the anti-tumor activity of chemotherapy agents commonly used in the treatment of B-cell NHL. We are continuing our research into defining the mechanisms by which OFA increases the lymphoma cell sensitivity threshold to chemotherapy agents and to novel target-specific small molecule inhibitors.