Two functional ligand binding sites on human CD30 separately located from the epitope of agonistic monoclonal antibodies (P6339)

Abstract

Abstract Human CD30 is a member of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF8). CD30 is considered to exist as homotrimers on the cell surface without binding to the ligand. Several anti-CD30 agonistic antibodies have been isolated. One of them is currently clinically used as an antibody drug conjugate for therapy of CD30-positive lymphomas. Despite progress in clinical application, the binding mode required for the agonistic activity of anti-CD30 antibodies has not been fully understood. In particular, the presence of two duplicated sequences including the TNF receptor domain in human CD30 complicates the analysis of binding-function relationships. In this study, we produced a series of recombinant conformation-carrying CD30 mutants whose TNF receptor domains and epitopes are replaced with corresponding sequences of CD40, based on the structures. We tested their ability to transduce signals for NFkB activation in reporter cells upon binding to CD30-ligand or agonistic antibodies. We found that both of the predicted ligand binding sites activate NFkB pathways by the binding of CD30-ligand-Fc fusion protein or CD30-ligand expressed on 293T cells. In addition, a group of agonistic antibodies appeared to bind an N-terminus site of the non-ligand binding site mutants and activated NFkB. Our results suggest that agonistic antibodies induce the active complex formation of membrane CD30 for potent signaling through a different binding mode than the natural ligand.