The Effects of Alemtuzumab (A) Dose-Sequence upon Rituximab (R)-Induced Reorganization of Lipid Raft Domains and In-Vitro/In Vivo Anti-Tumor Activity.

Following [R] binding, CD20 antigen redistributes into lipid raft domains (LRD) and initiates signaling events leading to apoptosis in malignant B-cells. In addition, the clustering of CD20 receptors into LRD results in a relative increase in the antigen density within a given area of the cell membrane, thus facilitating [R]-mediated complement mediated cytotoxicity (CMC) and/or antibody dependent cellular cytotoxicity (ADCC). Combinations of monoclonal antibodies (mAbs) directed against unique tumor-associated targets can potentially alter the reorganization of LRD and modify their anti-tumor activity. Our main objective was to study the effects of [A] upon [R]-mediated anti-tumor effects. For ADCC/CMC studies, ⁵¹Cr-labeled NHL cells (Raji, DHL-4, DHL-10, Karpas422 or Ramos cells) were exposed to concurrent [A+R], sequential [R→A] or [A→R] prior to the addition of peripheral blood mononuclear cells (effector:target ratio of 40:1) or human serum, respectively. Trastuzumab [I] served as isotype control. Following a 6-hour period incubation, supernatant was harvested and % lysis calculated. In addition, LRD of 1x10⁸ Raji cells exposed to similar mAb-combinations were extracted by sucrose gradient ultracentrifugation. Reorganization of CD20 into LRD was determined by Western blotting. For in vivo studies six to 8 week old SCID mice were inoculated by tail vein injection (iv) with 1x10⁶ Raji cells (day 0). After tumor engraftment (Day +7), animals were divided in seven cohorts to receive 8 doses of vehicle control, [R], [A], [I], alternating doses of [R+A], or sequential dosing of [A→R] or [R→A]. MAb were administered IV at 5mg/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. In vitro exposure to [A] prior to [R] therapy resulted a 30 to 50% decrease in rituximab-mediated ADCC. In addition, exposure of Raji cells to [A] led to a decrease in the amount of CD20 reorganized into LRD following rituximab exposure. No decrease in [R]-mediated ADCC or reorganization of CD20 into LRD was observed when [A] was administered following [R] therapy. In vivo studies demonstrated a better anti-tumor activity in animals treated with alternating doses of [R + A], when compared to [R→A], [A→R], [R] or [A] therapy. The median survival for mice treated with combination mAb therapy [R+A] was 90 days, compared to 43 and 70 days for those treated with [R→A] and [A→R] respectively. Combination therapy with [R+A] resulted in a statistically significant longer survival when compared to [R], [A], or [I] (P<0.009). In conclusion, alternating administration of [R] and [A] was more effective in controlling lymphoma growth than either mAb monotherapy or sequential administration of [R→A]/[A→R]. In addition, in vitro exposure of lymphoma cells to [A] prior to [R] resulted in decreased anti-tumor activity that may be explained by impairment in the polarization of CD20 into LRD. The sequence of administration of mAbs being used in combination mAb clinical trials may strongly influence the degree of total anti-tumor activity achieved.