Denosumab, a Fully Human Monoclonal Antibody to RANKL, Inhibits Bone Resorption and Increases BMD in Knock-In Mice That Express Chimeric (Murine/Human) RANKL

Abstract

RANKL is a TNF family member that mediates osteoclast formation, activation, and survival by activating RANK. The proresorptive effects of RANKL are prevented by binding to its soluble inhibitor osteoprotegerin (OPG). Recombinant human OPG-Fc recognizes RANKL from multiple species and reduced bone resorption and increased bone volume, density, and strength in a number of rodent models of bone disease. The clinical development of OPG-Fc was discontinued in favor of denosumab, a fully human monoclonal antibody that specifically inhibits primate RANKL. Direct binding assays showed that denosumab bound to human RANKL but not to murine RANKL, human TRAIL, or other human TNF family members. Denosumab did not suppress bone resorption in normal mice or rats but did prevent the resorptive response in mice challenged with a human RANKL fragment encoded primarily by the fifth exon of the RANKL gene. To create mice that were responsive to denosumab, knock-in technology was used to replace exon 5 from murine RANKL with its human ortholog. The resulting ''huRANKL'' mice exclusively express chimeric (human/murine) RANKL that was measurable with a human RANKL assay and that maintained bone resorption at slightly reduced levels versus wildtype controls. In young huRANKL mice, denosumab and OPG-Fc each reduced trabecular osteoclast surfaces by 95% and increased bone density and volume. In adult huRANKL mice, denosumab reduced bone resorption, increased cortical and cancellous bone mass, and improved trabecular microarchitecture. These huRANKL mice have potential utility for characterizing the activity of denosumab in a variety of murine bone disease models.

Prevention of glucocorticoid‐induced bone loss in mice by inhibition of RANKL

“…The hRANKL-knockin mice were generated by gene targeting (21). These hRANKLknockin mice carry the human instead of the murine exon 5 in their RANKL gene.…”

“…The resulting chimeric RANKL protein is recognized and inhibited by denosumab, a monoclonal antibody against hRANKL. Detailed analysis revealed that hRANKL-knockin mice have normal hematologic parameters, clinical blood chemistry, and histologic features of various organ systems, including the immune and cardiovascular systems (21).…”

“…Bone concentrations of RANKL and TRAP-5b were normalized to the total protein concentration as determined by a BCA Protein Assay (Pierce, Rockford, IL). The ratio of RANKL to OPG in bone extracts was expressed as the ratio of mRANKL to OPG in wild-type mice, and as the ratio of hRANKL to OPG in hRANKL-knockin mice, because chimeric RANKL is expressed under the control of the endogenous RANKL promoter in hRANKL-knockin mice and because the human RANKL assay more accurately reflects the circulating levels of chimeric RANKL than does the mRANKL assay (21).…”

“…In order to study the efficacy of specific RANKL inhibition in preclinical disease models, we recently generated human RANKL (hRANKL)-knockin mice (21). The rationale behind the generation of this mouse model is that the anti-human RANKL antibody denosumab is not reactive with murine RANKL (mRANKL) due to species specificity.…”

“…The rationale behind the generation of this mouse model is that the anti-human RANKL antibody denosumab is not reactive with murine RANKL (mRANKL) due to species specificity. Human RANKL-knockin mice carry the human instead of the murine exon 5 in their RANKL gene, and as a result express a chimeric RANKL protein that is capable of inducing bone resorption in mice while being fully inhibited by denosumab (21). Human RANKL-knockin mice represent the only small animal model for studying the safety and pharmacologic application of denosumab, which to date have included fracture repair (22) and ovariectomy-induced bone loss (23).…”