Abstract
The glycoprotein sclerostin has been identified as a negative regulator of bone growth. It exerts its function by interacting with the Wnt co-receptor LRP5/6, blocks the binding of Wnt factors and thereby inhibits Wnt signalling. Neutralizing anti-sclerostin antibodies are able to restore Wnt activity and enhance bone growth thereby presenting a new osteoanabolic therapy approach for diseases such as osteoporosis. We have generated various Fab antibodies against human and murine sclerostin using a phage display set-up. Biochemical analyses have identified one Fab developed against murine sclerostin, AbD09097 that efficiently neutralizes sclerostin's Wnt inhibitory activity. In vitro interaction analysis using sclerostin variants revealed that this neutralizing Fab binds to sclerostin's flexible second loop, which has been shown to harbour the LRP5/6 binding motif. Affinity maturation was then applied to AbD09097, providing a set of improved neutralizing Fab antibodies which particularly bind human sclerostin with enhanced affinity. Determining the crystal structure of AbD09097 provides first insights into how this antibody might recognize and neutralize sclerostin. Together with the structure–function relationship derived from affinity maturation these new data will foster the rational design of new and highly efficient anti-sclerostin antibodies for the therapy of bone loss diseases such as osteoporosis.
Highlights
Bone is not a dead tissue, but undergoes a permanent adaptation throughout life
15N-labelled murine sclerostin used for nuclear magnetic resonance (NMR) chemical shift titration mapping to determine the binding sites of Fab antibodies was produced in E. coli Rosetta (DE3) using M9 minimal medium supplemented with 0.5 g l21 15NH4Cl as described [30]
From the panning employing murine sclerostin four unique Fabs could be obtained, whereas panning using human sclerostin yielded seven unique Fabs. These 11 Fab fragments were expressed on a larger scale, purified and their binding properties were tested in an ELISA using murine and human sclerostin as well as unrelated control proteins to reveal their specificity
Summary
Bone is not a dead tissue, but undergoes a permanent adaptation throughout life. bone modelling occurs continuously to react to differences in mechanical load as well as physiological changes and is not just limited to remodelling after fracture repair. At the cellular level this is achieved by fine-tuning the activity of two ‘opposing’ cell types, the bone-forming osteoblasts and the bone-resorbing osteoclasts. Osteocytes, a third class of cells, act as master regulators controlling the activity of osteoblasts and osteoclasts through different hormones and signalling cascades. Disturbing this equilibrium will inevitably lead to pathological conditions. One such example is osteoporosis, which manifests itself by a low bone mineral density leading to a high fracture probability. The majority of therapeutic approaches against osteoporosis acted to prevent further bone loss usually by targeting the
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