Manipulation of receptor oligomerization as a strategy to inhibit signaling by TNF superfamily members

Abstract

Signaling by receptor activator of nuclear factor-κB (RANK) in response to its ligand RANKL, which is a member of the tumor necrosis factor (TNF) superfamily of cytokines, stimulates osteoclast formation and bone resorption. Thus, this ligand-receptor pair is a therapeutic target for various disorders, such as osteoporosis and skeletal metastasis. RANKL exists as a physiological homotrimer with each monomer recognizing a single molecule of RANK or the anti-osteoclastogenic decoy receptor osteoprotegerin (OPG). We engineered a RANKL protein in which all three monomers of RANKL were encoded as a single polypeptide chain, which enabled us to independently control receptor-binding at each binding interface. To generate an effective RANK inhibitor, we used an unbiased forward genetic approach to identify mutations in RANKL that had a 500-fold increased affinity for RANK, but had decreased affinity for the natural decoy receptor for RANKL, osteoprotegerin. Incorporating receptor-blocking mutations into this high-affinity RANKL variant generated a mutant RANKL that completely inhibited wild-type RANKL–induced osteoclastogenesis in vitro and bone resorption in mice. Our approach may be generalized to enable the inhibition of other TNF receptor signaling systems, which are implicated in wide range of pathological conditions.

Correlating RANK Ligand/RANK Binding Kinetics With Osteoclast Formation and Function

“…Constructs used for transient transfection of RANKL [Warren et al, ] or OPG [Nelson et al, ] were previously described. DNA for transfection was prepared using an endotoxin‐free DNA purification kit (Qiagen).…”

“…Most TNFSF members (including RANKL) exist as homotrimers in solution [Selmaj et al, ; Wu and Hymowitz, ] and it is generally assumed that the trimeric clustering of receptors is the primary initiating event for signal induction. We recently developed a novel inhibitor of RANKL‐induced osteoclast formation and function relying on the manipulation of receptor oligomerization [Warren et al, ]. In the process of engineering a single‐chain RANKL capable of antagonizing RANK signaling, we generated a panel of increased affinity RANKL mutants that allowed our construct to out‐compete WT RANKL binding.…”

Inflammatory osteolysis: a conspiracy against bone

“…In contrast to IL-17, IL-23, tions of anti-TNF-α therapy substantially reflect a failure to distinguish TNFR1 and TNFR2 (46,47). Recently developed strategies, including manipulation of affinities for the two receptors to promote selective binding and thus inhibition of TNFR1 while sparing TNFR2, may attenuate the problematic properties of TNF-α while maintaining those that are salutary (48).…”

Outflanking RANK with a Designer Antagonist Cytokine

“…Blocking TNF family cytokines is a successful therapeutic strategy in rheumatic and autoimmune diseases (2). Warren et al have generated a designer inhibitory cytokine against the TNF family member RANKL [receptor activator of nuclear factor κB (RANK) ligand] (3). Their method outlines a new strategy for inhibiting TNF family cytokines, and their fi ndings have interesting implications for the mechanism of signaling by RANK and related TNF family receptors.…”

“…Two interesting concepts have been integrated by Warren et al to make their cytokine antagonist (3). First, the authors generated a single-chain RANKL encoding three monomers of the cytokine linked by fl exible linkers.…”