Selective Modulation of Activated Protein C Activities by a Non-Active Site Targeting Nanobody Library.

Activated protein C (APC) is a pleiotropic coagulation protease with anticoagulant, anti-inflammatory, and cytoprotective activities. Selective modulation of these APC activities contributes to our understanding of the regulation of these physiological mechanisms and permits the development of therapeutics for the pathologies associated with these pathways. An antibody library targeting the non-active site of APC was generated with llama antibodies (nanobodies). Twenty-one nanobodies were identified that selectively recognize APC compared to the protein C (PC) zymogen. Overall, 3 clusters of nanobodies were identified based on the competition for APC in bio-layer interferometry studies. APC functional assays for anticoagulant activity, histone H3 cleavage, and PAR1 cleavage, were employed to understand their diversity. These functional assays revealed 13 novel nanobody-induced APC activity profiles via the selective modulation of APC pleiotropic activities, with the potential of regulating specific mechanisms for therapeutic purposes. Within these, 3 nanobodies (LP2, LP8, LP17) inhibited all 3 APC functions. Four nanobodies (LP1, LP5, LP16, LP20) inhibited only 2 of the 3 functions. Mono-function inhibition specific to APC anticoagulation activity was observed only by 2 nanobodies (LP9, LP11). LP11 was also found to shift the ratio of APC cleavage of PAR1 at R46 relative to R41 that results in APC-mediated biased PAR1 signaling and APC cytoprotective effects. Thus, LP11 has an activity profile that could potentially promote hemostasis and cytoprotection in bleedings associated with hemophilia or coagulopathy by selectively modulating APC anticoagulation and PAR1 cleavage profile.