Conjugation of Blocked Ricin to an Anti-CD19 Monoclonal Antibody Increases Antibody-Induced Cell Calcium Mobilization and CD19 Internalization

Abstract

AbstractCD19 (B4) is a signal transduction molecule restricted to the B-cell lineage and the target of the immunotoxin anti-B4–blocked ricin (anti-B4–bR), which is composed of the monoclonal antibody (MoAb) anti-B4 and the modified plant toxin blocked ricin. To explore the influence of conjugation of blocked ricin to anti-B4 on functional activation of CD19, we investigated the effects of anti-B4–bR, and that of unconjugated anti-B4, on intracellular calcium mobilization and ligand/receptor internalization. The data showed that anti-B4–bR was more potent than anti-B4 in triggering cell calcium mobilization. Two other immunotoxins that bind to the B-cell surface, anti-CD20–bR and anti-CD38–bR, were devoid of the calcium increasing effect of anti-B4–bR. Furthermore, anti-B4 conjugated to ricin A-chain was also without effect in Namalwa cells, indicating that the ricin B-chain component was required for anti-B4–bR effect. Anti-B4–bR-induced calcium mobilization was inhibited in the presence of lactose, yet the calcium response induced by cross-linking anti-B4–bR with a second step antibody was not affected. The extent of CD19 modulation induced by anti-B4–bR was higher than that induced by anti-B4, and lactose dampened the effect of the immunotoxin down to that of the MoAb. Moreover, the number of internalized immunotoxin molecules was higher than that of unconjugated MoAb. Although a mechanism involving dimerization of the immunotoxin cannot be excluded, our findings suggest that the residual binding activity of the blocked ricin B-chain to cell surface molecules plays an important role in the greater calcium fluxes and greater internalization rate of anti-B4–bR, and is of functional significance in the mechanism of intoxication of cells by the immunotoxin.