Multi-specific targeting of CD44 and the catalytic and hemopexin domains of MMP9 as a therapeutic strategy for osteoclast inhibition

Bone pathologies, such as osteoporosis, are characterized by excessive activity of osteoclasts (OCs)—bone-resorbing cells. As a therapeutic strategy to modulate this activity, we designed a chimera molecule that simultaneously inhibits two key regulators of OC function, matrix metalloproteinase 9 (MMP9) and CD44. The novel fusion molecule, designated C9-PEX, where C9 is a variant of tissue inhibitor of matrix metalloproteinase 2 (N-TIMP2) and PEX is the hemopexin domain in MMP9, exhibits strong binding affinity for CD44 and effectively inhibits the activity of the MMP9 catalytic domain, with an apparent low-picomolar Ki under our fluorogenic assay conditions. In addition, cell-binding assays showed enhanced binding of C9-PEX vs. comparative control proteins, C9 and Ala-C9-PEX. In murine bone marrow-derived OC cultures, gelatin zymography showed that C9-PEX significantly inhibited OC-secreted MMP9, whereas OC differentiation and bone-slice resorption assays showed reduced OC differentiation and reduced OC-mediated bone resorption without detectable toxic effects on cell proliferation and survival. At matched nominal concentrations, C9-PEX consistently outperformed the comparative control proteins and a 1:1 C9+PEX mixture, supporting a functional advantage of simultaneous target engagement within a single fusion format. Although binding-site/valency-equivalent dosing, formal combination quantification, and normalization of resorption area to OC number on bone slices were not applied in the present study, these findings support C9-PEX as a promising multi-specific inhibitor of OC-mediated resorption in a murine in vitro system. Future studies using human OCs and in vivo models will be required to define the translational efficacy of C9-PEX and the exposure levels required for therapeutic activity. • C9-PEX fusion protein simultaneously targets membrane-bound and secreted proteins • Bi-specific C9-PEX demonstrates synergistic mechanism of osteoclast inhibition • C9-PEX demonstrates superior efficacy vs. monospecific controls and their mixture