Generation of antagonistic biparatopic anti-CD30 antibody from an agonistic antibody by precise epitope determination and utilization of structural characteristics of CD30 molecule

Abstract

Listen

Translate article

Abstract Background CD30 is a member of the tumor necrosis factor receptor superfamily. Recently, blocking CD30-dependent intracellular signaling has emerged as potential strategy for immunological regulation. Development of antibody-based CD30 antagonists is therefore of significant interest. However, a key challenge is that the bivalent form of natural antibody can crosslink CD30 molecules, leading to signal transduction even in the absence of specific ligand, CD153. Biparatopic antibodies (BpAbs) offer a solution, using two different variable fragments (Fvs) to bind distinct epitopes on a single antigen molecule. BpAbs format is an attractive alternative of natural antibody by potentially avoiding unwanted crosslinking and signaling induction. Methods We systematically characterized 36 BpAbs, each designed with pairs of Fvs binding to nine distinct epitopes across the CD30 extracellular domain. We first identified the precise epitope sites of the nine antibodies by assessing the binding to multiple orthologous CD30 proteins and mutants. We then produced the 36 BpAbs and analyzed their biological activities and binding modes. Results Among 36 BpAbs, we identified both potent ligand-independent agonists and ligand-blocking antagonists, with many displayed reduced signal activation, including 1:1-binding antagonists derived from AC10, a strong agonist developed for lymphoma therapy. Epitope dependency in reduced signaling activity was observed and associated with the flexible nature of CD30 protein. Conclusions We successfully developed antagonistic BpAbs against CD30 by controlling the stoichiometry of antibody–antigen binding mode. This study elucidated the mechanism of signaling induction, informing the design strategies of the development of biparatopic antibodies. Statement of Significance Thirty-six bivalent biparatopic antibodies targeting pairs of nine distinct topographical epitopes distributed over CD30 were developed. These BpAbs exhibited a range of signaling activities from agonism to antagonism, with many displaying reduced signal activation. Notably, 1:1-binding antagonists were developed with no signal induction. The mechanism was elucidated by epitope information.