Switchbody, an Antigen‐Responsive Enzyme Switch Based on Antibody and Its Working Principle

This paper presents an antibody‐based enzyme switch, termed a “Switchbody”, which is fused with an enzyme‐derived fragment to the N‐terminus of an antibody. As its working principle, the fused fragment is trapped within the antibody and released upon antigen binding, leading to enzyme reconstitution and a subsequent increase in enzyme activity. This “Trap and Release” principle provides a framework for designing next‐generation enzyme switches. Abstract An enzyme switch, termed “Switchbody”, is developed by fusing an antibody with a fragment of a split enzyme for the precise regulation of enzyme activity in response to an antigen. A luciferase‐based Switchbody is engineered by fusing the NanoLuc luciferase fragment HiBiT to the N‐terminus of an antibody. The enzyme activity of the Switchbody increases upon the addition of an antigen in a dose‐dependent manner in the presence of the complementary fragment LgBiT and its substrate furimazine, demonstrating the potential of the luciferase‐based Switchbody as a biosensor. As its working principle, ELISA shows that the interaction between HiBiT and LgBiT is facilitated by antigen binding. Moreover, X‐ray crystallography and NMR reveal the heterogeneous trapped state of the HiBiT region and an increasing motility of HiBiT region upon antigen binding, respectively. MD simulations and luminescence measurements show that antigen disrupted the trapping of HiBiT in the antibody, enabling its release. By applying this “Trap and Release” principle to Protein M, an antibody‐binding protein, label‐free IgG antibodies are successfully converted into bioluminescent Switchbodies. This adaptable Switchbody platform has the potential to expand switching technology beyond luciferase to various other enzymes in the future. This paper presents an antibody-based enzyme switch, termed a “Switchbody”, which is fused with an enzyme-derived fragment to the N-terminus of an antibody. As its working principle, the fused fragment is trapped within the antibody and released upon antigen binding, leading to enzyme reconstitution and a subsequent increase in enzyme activity. This “Trap and Release” principle provides a framework for designing next-generation enzyme switches. Abstract An enzyme switch, termed “Switchbody”, is developed by fusing an antibody with a fragment of a split enzyme for the precise regulation of enzyme activity in response to an antigen. A luciferase-based Switchbody is engineered by fusing the NanoLuc luciferase fragment HiBiT to the N-terminus of an antibody. The enzyme activity of the Switchbody increases upon the addition of an antigen in a dose-dependent manner in the presence of the complementary fragment LgBiT and its substrate furimazine, demonstrating the potential of the luciferase-based Switchbody as a biosensor. As its working principle, ELISA shows that the interaction between HiBiT and LgBiT is facilitated by antigen binding. Moreover, X-ray crystallography and NMR reveal the heterogeneous trapped state of the HiBiT region and an increasing motility of HiBiT region upon antigen binding, respectively. MD simulations and luminescence measurements show that antigen disrupted the trapping of HiBiT in the antibody, enabling its release. By applying this “Trap and Release” principle to Protein M, an antibody-binding protein, label-free IgG antibodies are successfully converted into bioluminescent Switchbodies. This adaptable Switchbody platform has the potential to expand switching technology beyond luciferase to various other enzymes in the future. Advanced Science, Volume 12, Issue 44, November 27, 2025.