An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling

The transforming growth factor beta isoforms, TGF-beta 1, -beta 2, and -beta 3, are small secreted homodimeric signaling proteins with essential roles in regulating the adaptive immune system and maintaining the extracellular matrix. However, dysregulation of the TGF-beta pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-beta s in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF-beta monomer, lacking the heel helix, a structural motif essential for binding the TGF-beta type I receptor (T beta RI) but dispensable for binding the other receptor required for TGF-beta signaling, the TGF-beta type II receptor (T beta RII), as an alternative therapeutic modality for blocking TGF-beta signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF-beta monomers and bound T beta RII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF-beta signaling with a K-i of 20-70 nM. Investigation of the mechanism showed that the high affinity of the engineered monomer for T beta RII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit T beta RI, enabled it to bind endogenous T beta RII but prevented it from binding and recruiting T beta RI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF-beta signaling and may inform similar modifications of other TGF-beta family members.