TRAIL-R2 Superoligomerization Induced by Human Monoclonal Agonistic Antibody KMTR2

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.

Antibody Heavy Chain Variable Domains of Different Germline Gene Origins Diversify through Different Paths

“…Possibly R80 is less important for maintenance of the integrity of the domain or the general architecture of the binding site in antibodies derived from this germline gene. Interestingly, reorientation of R80 has been demonstrated in one antibody ( 24 ) with a likely origin in IGHV1-8. This was also associated with a reorientation of CDRH2 as determined by X-ray crystallography (Figures 14 E-H).…”

“…Sequence variability was calculated as the number of amino acids encoded by more than 1% of all reads, divided by the fraction of reads encoding the most common residue. For comparison with real protein structures, examples of structures with an origin in IGHV1-18 (PDB: 3SDY) ( 22 ), IGHV1-8 (PDB 3X3G and 3U1S) ( 23 , 24 ), IGHV2-5 (PDB: 3QRG), IGHV3 subgroup (PDB: 2R56 and 3FZU) ( 25 , 26 ), IGHV4-39 (PDB: 5C6T) ( 27 ), IGHV4-59 (PDB: 3HI1) ( 28 ), and IGHV5-51 (PDB: 4BUH) ( 29 ) were identified using the IMGT/3Dstructure-DB web interface ( 30 ), and coordinates were downloaded from RCSB Protein Data Bank. 2 Structures were visualized using MacPyMOL v1.8.0.6.…”

Higher-Order Clustering of the Transmembrane Anchor of DR5 Drives Signaling

“…Additionally, mutations in the PLAD away from the ligand-binding site have been found to affect ligand binding, suggesting an allosteric coupling between ligand binding and PLAD association (Chan et al, 2000). There is also a tendency for agonistic antibodies to bind to the CRD1 and CRD2 regions of TNFRSF members that overlap with the ECD PLAD (Chodorge et al, 2012;Siegel et al, 2000b;Tamada et al, 2015), suggesting that breaking the PLAD interaction contributes to the agonistic phenotypes. Interestingly, O-linked glycosylation of DR5 ECD could augment ligand-induced receptor signaling (Wagner et al, 2007), which could also be explained by weakening of the PLAD interaction by O-Glycan modification Figure 6.…”

Crystal structure of CD27 in complex with a neutralizing noncompeting antibody

“…The orientation of Fab inline with the receptor axis is unique among anti-TNFR superfamily antibodies that have been structurally characterized. Typically, they bind around CRD1 and CRD2 on the side of the receptor molecule (Fellouse et al, 2005;Li et al, 2006;Adams et al, 2008;Chodorge et al, 2012;Graves et al, 2014;Tamada et al, 2015;Yamniuk et al, 2016). The location of the epitope at the top edge provides an easier access for an antibody, which may be beneficial, particularly in the tumor microenvironment.…”