A novel agonistic antibody to human death receptor 4 induces apoptotic cell death in various tumor cells without cytotoxicity in hepatocytes

Abstract

The proapoptotic tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptors death receptor (DR) 4 and DR5 are attractive targets to develop the receptorspecific agonistic monoclonal antibodies (mAb) as anticancer agents because of their tumor-selective cell death-inducing activity. Here, we report a novel agonistic mAb, AY4, raised against human DR4 in mice. ELISA analysis revealed that AY4 specifically bound to DR4 without competition with TRAIL for the binding. Despite distinct binding regions of AY4 on DR4 from those of TRAIL, AY4 as a single agent induced caspase-dependent apoptotic cell death of several tumor types through the extrinsic and/or intrinsic pathways without substantial cytotoxicity to normal human hepatocytes. Further, the AY4-sensitive cells followed the same cell death characteristics classified as type I and type II cells by the response to TRAIL, suggesting that the cell death profiles in responses to DR4 and/or DR5 stimulation are determined by the downstream signaling of the receptor rather than the kind of receptor. Noticeably, AY4 efficiently induced cell death of Jurkat cells, which have been reported to be resistant to other anti-DR4 agonistic mAbs, most likely due to the unique epitope property of AY4. In vivo administration of AY4 significantly inhibited tumor growth of human non-small cell lung carcinoma preestablished in athymic nude mice. Conclusively, our results provide further insight into the DR4-mediated cell death signaling and potential use of AY4 mAb as an anticancer therapeutic agent, particularly for

Histone deacetylase inhibitors synergistically potentiate death receptor 4-mediated apoptotic cell death of human T-cell acute lymphoblastic leukemia cells

“…Soluble TRAIL (residues 114-281 with a C-terminal 69 His-tag; described as TRAIL hereafter) and mAb AY4 were prepared as previously described [28]. SAHA, doxorubicin, and etoposide were from Sigma (St Louis, MO); VPA and MG132 were from Calbiochem (San Diego, CA); SN50 was from BIOMOL (Plymouth Meeting, PA).…”

“…The results are presented as mean ± standard deviation (SD) values from at least three independent experiments. The cytotoxicity data of chemotherapeutic agents were analyzed by using a four parameter sigmoidal curve fit on Sigma plot software (SPSS Inc.) to calculate IC 50 , the concentration of an anti-cancer agent giving a 50% reduction in cell viability [28,31]. The IC 50 values are presented as mean ± SD of triplicate determinations (Supplementary Table S1).…”

“…A standard procedure for Western blotting was performed as described previously [28,32]. The horseradish peroxidase conjugated anti-rabbit or anti-mouse IgG was used to visualize proteins via an enhanced chemiluminescence system (ECL, Thermo Scientific Inc, Rockford, IL).…”

“…We have recently reported the development of a murine mAb, AY4 (IgG1, j), which binds specifically to the extracellular domain of DR4 and induces caspase-dependent apoptotic cell death in various types of cancer cells without significant toxicity to normal human hepatocytes [28]. Although AY4 recognizes regions of DR4 distinct from those bound by TRAIL, AY4 stimulation of DR4 can activate both the extrinsic and intrinsic apoptotic pathways in AY4-sensitive tumor cells [28,29].…”

“…Although AY4 recognizes regions of DR4 distinct from those bound by TRAIL, AY4 stimulation of DR4 can activate both the extrinsic and intrinsic apoptotic pathways in AY4-sensitive tumor cells [28,29]. In this study, we further investigate the specific role of DR4 in inducing apoptosis by studying the susceptibility of various leukemia cell lines to AY4 alone or in combination with various chemotherapeutic agents, in comparison with TRAIL.…”