ADR3, a next generation i-body to human RANKL, inhibits osteoclast formation and bone resorption

Osteoporosis is a chronic skeletal condition characterized by low bone mass and deteriorated microarchitecture of bone tissue and puts tens of millions of people at high risk of fractures. New therapeutic agents like i-bodies, a class of next-generation sin-gle-domain antibodies, are needed to overcome some limitations of conventional treatments. An i-body is a human immuno-globulin scaffold with two long binding loops that mimic the shape and position of those found in shark antibodies, the var-iable new antigen receptors of sharks. Its small size (-12 kDa) and long binding loops provide access to drug targets, which are considered undruggable by traditional monoclonal antibodies. Here, we have successfully identified a human receptor activator of nuclear factor-kappa B ligand (RANKL) i-body, ADR3, which demonstrates a high binding affinity to human RANKL (hRANKL) with no adverse effect on the survival or proliferation of bone marrow-derived macrophages. Differential scanning fluorimetry suggested that ADR3 is stable and able to tolerate a wide range of physical environments (including both tempera-ture and pH). In addition, in vitro studies showed a dose-dependent inhibitory effect of ADR3 on osteoclast differentia-tion, podosome belt formation, and bone resorption activity. Further investigation on the mechanism of action of ADR3 revealed that it can inhibit hRANKL-mediated signaling path-ways, supporting the in vitro functional observations. These clues collectively indicate that hRANKL antagonist ADR3 at-tenuates osteoclast differentiation and bone resorption, with the potential to serve as a novel therapeutic to protect against bone loss.