Takafumi Tomura

Enhanced Apoptosis and Tumor Regression Induced by a Direct Agonist Antibody to Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Receptor 2

Purpose: Substantial evidence indicates that supraoligomerization of the death receptors for Fas ligand and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is necessary for efficient activation of the apoptotic pathway. Bivalent IgG antibodies can induce the efficient apoptosis by mimicking the natural ligands but only after these antibodies are further oligomerized by cross-linking. In this study, we generated a novel agonist antibody to TRAIL receptor 2 (TRAIL-R2) capable of inducing apoptosis without cross-linking and elucidated its mode of action and efficacy. Experimental Design: A fully human antibody to TRAIL-R2, KMTR2, was generated from KM Mouse immunized with TRAIL-R2 ectodomain. Apoptosis-inducing activities of unfractionated or purified monomeric IgG of KMTR2 was evaluated in the presence or absence of cross-linkers, secondary antibodies or Fc receptor–expressing effector cells, against human colorectal adenocarcinoma Colo205. Oligomerization of TRAIL-R2 was analyzed by size exclusion chromatography and confocal microscopy, and in vivo efficacy was examined in Colo205 xenograft model. Results: KMTR2 specifically recognized TRAIL-R2 and induced apoptosis with or without cross-linking. Size exclusion chromatography showed that the apoptosis activity coeluted with monomeric IgG and was effective independent of secondary antibody or Fc receptor–expressing effector cells. The antibody formed supracomplexes with soluble recombinant and membrane-anchored TRAIL-R2 and enhanced clustering of TRAIL-R2 on cell surface without cross-linking. KMTR2 was dramatically efficacious in reducing established human tumor. Conclusion: Our findings indicate that novel agonist antibody KMTR2 can direct antibody-dependent oligomerization of TRAIL-R2 and initiates efficient apoptotic signaling and tumor regression independent of host effector function. Thus, the direct agonist would be a lead candidate for cancer therapeutics.

The crystallographic analysis of glycosylation-inhibiting factor

Publisher Summary Glycosylation-inhibiting factor (GIF) inhibits N-glycosylation of IgE-binding factors. The unglycosylated IgE-binding factor then selectively suppresses IgE synthesis. Further, GIF appears to be a subunit of antigenspecific suppressor T cell factors that facilitate the generation of antigen-specific suppressor T cells. Recent studies indicate that post-translational modification of GIF in suppressor T cells is required for the generation of the biological activity. However, the relationship between GIF bioactivity and the conformational transition of the protein is not known. To understand the mechanisms of GIF functions, this chapter studies the crystal structure of recombinant human GIF. It is apparent that GIF has a novel tertiary structure. The chapter discusses the crystallographic analysis of GIF. The overall structure of GIF trimer is found to be a three-fold-related barrel structure, which is composed of three six-stranded β-sheets on the inside and six α-helices on the outside. Each subunit consists of two β-α-α motifs related by a pseudo-twofold axis. The trimer structure is formed by intermonomer hydrogen bonds and hydrophobic interfaces among β-sheets. There is a 5-A diameter “hole” through the middle of the barrel. The barrel structure of GIF in part resembles “trefoil” cytokines, such as interleukin-1 and fibroblast growth factor.