Negative modulation of B-cell function via inhibitory receptor signaling by using novel diabody-based Dual-Affinity Re-Targeting (DART) molecules

Abstract The Fc receptor, CD32B, functions as an inhibitory receptor and, when co-ligated with the B-cell antigen receptor (BCR) complex, dampens signaling and immune activation. Ligation of activating and inhibitory receptors is an obligatory event for CD32B inhibition coupling in B cells, which heightens the need for a bispecific molecule for interventional purposes. To exploit this mechanism, we have engineered a novel and stable bispecific diabody scaffold that facilitates heterodimeric assembly with high predictability. This Dual-Affinity Re-Targeting (DART) scaffold was used to couple an Fv specific for the inhibitory receptor, CD32B, with an Fv specific for the Ig-beta component (CD79B) of the BCR. The resulting CD32BxCD79B DART molecule was capable of co-ligating the activating and the inhibitory receptors on the B-cell surface, resulting in reduced BCR-induced calcium mobilization and phosphorylation of selected signaling molecules. These biochemical changes correlated with inhibition of B-cell proliferation and Ig secretion upon B-cell activation. In addition, a DART protein specific for the mouse orthologs of CD32B and CD79B reduced disease severity in a collagen-induced arthritis model. This novel approach to B-cell function modulation forms the basis for translating inhibitory signaling into therapeutic tools for managing autoimmune and inflammatory diseases.