Abstract
The fully human monoclonal antibody KMTR2 acts as a strong direct agonist for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2 (TRAIL-R2), which is capable of inducing apoptotic cell death without cross-linking. To investigate the mechanism of direct agonistic activity induced by KMTR2, the crystal structure of the extracellular region of TRAIL-R2 and a Fab fragment derived from KMTR2 (KMTR2-Fab) was determined to 2.1 Å resolution. Two KMTR2-Fabs assembled with the complementarity-determining region 2 of the light chain via two-fold crystallographic symmetry, suggesting that the KMTR2-Fab assembly tended to enhance TRAIL-R2 oligomerization. A single mutation at Asn53 to Arg located at the two-fold interface in the KMTR2 resulted in a loss of its apoptotic activity, although it retained its antigen-binding activity. These results indicate that the strong agonistic activity, such as apoptotic signaling and tumor regression, induced by KMTR2 is attributed to TRAIL-R2 superoligomerization induced by the interdimerization of KMTR2.
Highlights
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L)[1,2] induces apoptosis in a wide variety of human cancer cell lines, but not in most normal human cells[3,4]
The tertiary structure of ecTRAIL-R2 in complex with KMTR2-Fab was determined to 2.1 Å resolution by X-ray crystallography (Fig. 1a)
Our results suggested that the 2:2 configuration shown in Fig. 2a was an essential unit for sufficient KMTR2 direct agonistic activity
Summary
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L)[1,2] induces apoptosis in a wide variety of human cancer cell lines, but not in most normal human cells[3,4]. The structure of the extracellular region of TRAIL-R2 (ecTRAIL-R2) in complex with TRAIL has shown that similar to other members of the TNF and TNF receptor superfamily, a TRAIL trimer binds to three receptors[11,12,13], suggesting that a trimeric ligand–receptor complex is the functional unit for signaling[14] This trimeric complex has been determined for ternary (3:3:3) complex with Fab fragment derived from AMG 655 which increases antitumor activity in cooperation with TRAIL15. Based on its crystallographic symmetry, the dimeric complex structure of KMTR2 appeared to be a functional unit for generating a superoligomeric complex Interference of this interface with an Asn[53] to Arg mutation in the light kappa chain of KMTR2 resulted in the loss of the direct agonistic activity but not the binding activity of KMTR2, indicating that interdimerization of KMTR2 is responsible for TRAIL-R2 superoligomerization, leading to the strong agonistic activity of KMTR2
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