Tomoko Ise

CD30 is a type I membrane protein that has been successfully targeted for lymphoma therapy using Brentuximab vedotin, an antibody-drug conjugate. Recently, the potential of blocking CD30-dependent NF-κB intracellular signaling has gained attention for treating inflammatory disorders. Development of antibody-based CD30 antagonists would broaden therapeutic strategies. A challenge in developing antagonistic antibodies is that the bivalent form of natural antibody format inevitably cross-links trace amounts of CD30 molecules, leading to signal transduction. In this study, we developed a series of biparatopic antibodies with each pair of antibody variable domains (Fvs) binding to distinct epitopes on CD30, and evaluated their biological activities and binding modes. Initially, we precisely identified epitope sites of the nine antibodies precisely by assessing binding to multiple orthologous CD30 proteins and mutants. We then produced 36 biparatopic antibodies covering all possible combinations of the nine Fvs, and analyzed their biological activities. Among these, we identified both potent agonists and antagonists. Notably, a significant proportion of the biparatopic antibodies displayed reduced agonistic activities, including 1:1-binding antagonists derived from a Fv of a strong agonist previously developed for lymphoma therapy, AC10. The mechanism of signaling activity induction is discussed using epitope information, which leads to the strategies of the development of biparatopic antibodies.

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.