In Vitro Synergistic Anti-Tumor Activity by Targeting TRAIL-R1 and CD20 Antigen by Combining HGS-ETR1 (Agonistic Human Monoclonal Antibody to TRAIL Receptor 1) and Rituximab Monoclonal Antibody Against Non-Hodgkin’s Lymphoma Cells (NHL).

Tumor necrosis factor-related apoptosis ligand (TRAIL) or agonist antibodies targeting specific TRAIL-receptors possess significant in vitro and in vivo anti-tumor activity. HGS-ETR1 (mapatumumab) and HGS-ETR2 are two fully human recombinant, high affinity IgG1λ mAbs targeting TRAIL-R1 (Death-receptor 4, DR-4) and TRAIL-R2 (Death-receptor 5, DR-5), respectively. TRAIL-receptor agonist mAbs trigger the extrinsic (mitochondria-independent) apoptotic pathway and enhance the anti-tumor effects of chemotherapy drugs against NHL. Combining TRAIL-receptor agonist mAbs with biological agents (e.g. rituximab) capable of activating the intrinsic (mitochondria-dependent) apoptotic pathway may result in synergistic effects against NHL.

Objective: To study the biological effects of rituximab when combined with either HGS-ETR1 or HGS-ETR2.

Materials and methods: A panel of NHL cell lines [Raji, SUDHL-4, SU-DHL-10, Ramos, and two rituximab resistant cell lines (RRCL), 2R and 4RH] were utilized. Expression of CD20, TRAIL-R1 and TRAIL-R2 in the NHL cells used were evaluated by flow cytometry. NHL cells were exposed to HGS-ETR1 or HGS-ETR2 followed by either rituximab, istoype control antibody or media alone. DNA synthesis and cell growth arrest were quantified by [H3] Thymidine incorporation assays at 24 and 48 hrs. Induction of apoptosis was detected by multiparameter flow cytometric analysis. For antibody-dependent cellular cytotoxicity (ADCC)/Complement mediated cytotoxicity (CMC) studies, ⁵¹Cr-labeled NHL cells were exposed to HGS-ETR1 or HGS-ETR2 (5μg/ml) +/− rituximab or isotype control antibody (10μg/ml) and peripheral blood mononuclear cells (Effector: Target ratio 40:1) or human serum, respectively. ⁵¹Cr-release was measured and the percentage of lysis was calculated. Statistical analysis of results was performed using the Chi-square test. Results: In sensitive cells in vitro exposure to HGS-ETR1 resulted in significant apoptosis (30–50%) and decrease in DNA synthesis. The combination of HGS-ETR1 and rituximab resulted in significant inhibition of cell proliferation (90% reduction) when compared to either HGS-ETR1 (60% reduction) or rituximab (5% reduction) alone at 24 and 48 hrs. In sensitive cells HGS-ETR1 induced CMC and ADCC as a single agent. No significant anti-tumor activity was observed with HGS-ETR2. The biological activity of anti-TRAIL receptors mAbs did not correlate to TRAIL-R1 or TRAIL-R2 cell surface antigen expression. Conclusions: Targeting TRAIL-R1 with HGS-ETR1 induces apoptosis, cell growth arrest and in some cell lines ADCC/CMC. The combination of HGS-ETR1 with rituximab results in significant synergistic activity (i.e. anti-proliferation) and warrants further pre-clinical and clinical evaluation in B-cell lymphomas.