Antagonist nanobodies prevent protease inhibition by CD109

Cluster of differentiation 109 (CD109) is a cell surface protein found on many human cell types, including keratinocytes, platelets, and T-cells. It is a protease inhibitor of the α-macroglobulin protein superfamily and is reported to affect signaling pathways such as TGFβ and STAT3. Here, we characterized two CD109-specific nanobodies, E1 and B3, that block CD109's interaction with proteases through distinct mechanisms. E1 binds to an epitope on the thiol ester domain of CD109 with high affinity (⁓10^-9 M K_D), preventing its conjugation to proteases. B3 binds to an epitope near the MG3, MG4, and bait region of CD109 with moderate affinity (⁓10^-7 M K_D), preventing bait region cleavage. Both mechanisms prevent protease inhibition by CD109. The nanobodies retained their functionalities as Fc fusions, and treatment with B3-Fc prevented the release of membrane-bound CD109 from HEK293F cells by endogenous proteases. These nanobodies provide valuable new tools for investigating CD109's interactions with proteases and may have therapeutic applications in diseases where CD109 plays a pathogenic role.