Kentaro Nishiyama

Template reactions are site‐specific, rapid chemical reactions driven by high‐affinity biomolecular interactions. Recently, our group reported a reaction triggered by antigen–antibody interactions, named the biepitopic antigen‐templated chemical reaction (BATER). In BATER, reactive groups are conjugated to two antibodies recognizing different epitopes of the same antigen. Upon antigen binding, these groups are brought into proximity, enabling rapid reaction. However, the report was limited to tumor necrosis factor receptor 2, and its applicability to other targets remained unclear. In this study, we report strategies to expand BATER to antigens with diverse structural characteristics. Three distinct approaches were applied: (1) epitope selection leveraging structural flexibility of the antigen; (2) the use of protein domains as spacers; and (3) the use of small engineered antibodies. These strategies enabled BATER with CD30 and human epidermal growth factor receptor 2, demonstrating its broad applicability. The expanded range of antigens targeted by BATER will pave the way for potential applications in chemical biology, diagnosis, and therapy.

Conventional bivalent antibodies against cell surface receptors often initiate unwanted signal transduction by crosslinking two antigen molecules. Biparatopic antibodies (BpAbs) bind to two different epitopes on the same antigen, thus altering crosslinking ability. In this study, we develop BpAbs against tumor necrosis factor receptor 2 (TNFR2), which is an attractive immune checkpoint target. Using different pairs of antibody variable regions specific to topographically distinct TNFR2 epitopes, we successfully regulate the size of BpAb–TNFR2 immunocomplexes to result in controlled agonistic activities. Our series of results indicate that the relative positions of the two epitopes recognized by the BpAb are critical for controlling its signaling activity. One particular antagonist, Bp109-92, binds TNFR2 in a 1:1 manner without unwanted signal transduction, and its structural basis is determined using cryo-electron microscopy. This antagonist suppresses the proliferation of regulatory T cells expressing TNFR2. Therefore, the BpAb format would be useful in designing specific and distinct antibody functions.

Proximity‐induced chemical reactions are site‐specific and rapid by taking advantage of their high affinity and highly selective interactions with the template. However, reactions induced solely by antibody–antigen interactions have not been developed. Herein, we propose a biepitopic antigen‐templated chemical reaction (BATER) as a novel template reaction. In BATER, reactive functional groups are conjugated to two antibodies that interact with two epitopes of the same antigen to accelerate the reaction. We developed a method for visualizing the progress of BATER using fluorogenic click chemistry for optimal antibody selection and linker design. The reaction is accelerated in the presence of a specific antigen in a linker length‐dependent manner. The choice of the antibody epitope is important for a rapid reaction. This design will lead to various applications of BATER in living systems.

Proximity‐induced chemical reactions are site‐specific and rapid by taking advantage of their high affinity and highly selective interactions with the template. However, reactions induced solely by antibody–antigen interactions have not been developed. Herein, we propose a biepitopic antigen‐templated chemical reaction (BATER) as a novel template reaction. In BATER, reactive functional groups are conjugated to two antibodies that interact with two epitopes of the same antigen to accelerate the reaction. We developed a method for visualizing the progress of BATER using fluorogenic click chemistry for optimal antibody selection and linker design. The reaction is accelerated in the presence of a specific antigen in a linker length‐dependent manner. The choice of the antibody epitope is important for a rapid reaction. This design will lead to various applications of BATER in living systems.

Conventional bivalent antibodies against cell surface receptors often initiate unwanted signal transduction by crosslinking two antigen molecules. Biparatopic antibodies (BpAbs) bind to two different epitopes on the same antigen, thus altering crosslinking ability. In this study, we developed BpAbs against tumor necrosis factor receptor 2 (TNFR2), which is an attractive immune checkpoint target. Using different pairs of variable regions specific to topographically distinct TNFR2 epitopes, we successfully regulated the size of BpAb-TNFR2 immunocomplexes to result in controlled agonistic activities. One particular antagonist BpAb bound TNFR2 in 1:1 ratio without unwanted signal transduction, with its structural basis revealed by cryo-electron microscopy. This antagonist suppressed the proliferation of regulatory T cells expressing TNFR2. Therefore, the BpAb format would be useful in designing specific and distinct antibody functions.