Identification and characterization of nanobodies specific for the human ubiquitin-like ISG15 protein

Interferon-induced ubiquitin (Ub)-like modifier Interferon Stimulated Gene 15 (ISG15) functions both intracellularly and as a secreted protein with cytokine-like properties. The ISG15 pathway is implicated in various diseases, including cancer and inflammatory disorders, but understanding its precise roles has been challenging because of limited availability of tools to study ISG15 biology. Here, we report the development of two novel nanobodies that target human ISG15, obtained through alpaca immunization and phage display. These nanobodies, VHH_ISG15-A and VHH_ISG15-B, exhibit nanomolar binding affinities and recognize distinct epitopes on ISG15's C- and N-terminal domains, respectively, as demonstrated by NMR and X-ray structural analyses. Both nanobodies enable the immunoprecipitation and proteomic identification of ISGylated substrates with minimal background contamination. VHH_ISG15-A is compatible with immunoblotting and recognizes unconjugated ISG15 under denaturing conditions. Functional assays showed that VHH_ISG15-A, but not VHH_ISG15-B, inhibits ubiquitin-specific peptidase 16-mediated deISGylation, likely by steric hindrance at the ISG15-binding interface. These results underscore the utility of VHH_ISG15-A and VHH_ISG15-B as tools to study ISG15 biology.