ALTERATION OF B-CELL ANTIGEN RECEPTOR SIGNALING BY CD19 CO-LIGATION -A STUDY WITH BISPECIFIC ANTIBODIES

The activation of B cell antigen receptor-associated protein tyrosine kinases is an early and crucial event in B-cell signaling. Apart from the B-cell antigen receptor (BCR), the B cell-specific transmembrane g lycoprotein CD19 has also been shown to directly activate intracellula r signaling cascades. In addition, because CD19 and the BCR are associ ated on the surface of activated B-cells, it has been proposed that cl ose approximation between these two entities is crucial for optimal B- cell triggering. To test this hypothesis, bispecific antibodies were g enerated that bind membrane IgM and CD19 simultaneously, Although CD19 bispecific antibodies strongly induced tyrosine phosphorylation, they were, in contrast to mu F(ab)(2) fragments, unable to induce a prolif erative response. Detailed analysis of the early signaling events show ed that compared with mu F(ab)(2) fragments CD19 bispecific antibodies potently raised the intracellular [Ca2+], which was correlated with a n efficient tyrosine phosphorylation of syk. Strikingly, the assembly of Grb2 complexes that may couple the BCR to p21(ras) was clearly alte red by the CD19 bispecific antibody. In addition to the reported She a nd 145-kDa phosphoproteins, a prominent 90-95-kDa phosphoprotein resem bling CD19 was detected in the Grb2 complexes. Thus, studies with CD19 bispecific antibodies show that CD19 co-ligation both quantitatively and qualitatively alters BCR signaling.