Construction of optimized bispecific antibodies for selective activation of the death receptor CD95

We have previously reported that bispecific antibodies directed to different target antigens on lymphoma cells and to the death receptor CD95/Fas/Apo-1 selectively kill these cells, thus providing an attractive strategy for the selective stimulation of CD95 on the surface of tumor cells. Here, we further explore the general applicability of this approach under more stringent conditions using various bispecific antibodies directed to different target antigens on glioblastoma cells which express relatively low levels of CD95. We found that bispecific CD95 antibodies targeting the neuronal glial antigen-2 induce CD95-mediated apoptosis selectively in glioblastoma cells expressing this target antigen. A recombinant bispecific single-chain antibody was as effective as a chemically hybridized F(ab')(2) fragment with identical specificities. In contrast, a bispecific F(ab')(2) fragment binding to the epidermal growth factor receptor on the glioblastoma cells failed to induce apoptosis. This is most likely due to the exclusively unicellular binding of this particular fragment to target cells expressing both the epidermal growth factor receptor and CD95. If this type of binding in a cis configuration is favored by a particular bispecific antibody, rather than a bicellular binding in trans, effective cross-linking of CD95 does not occur and apoptosis is not induced. To facilitate bicellular binding in a trans configuration, we constructed a bispecific antibody directed to the extracellular matrix protein tenascin. As expected, this reagent was the most effective of all the antibodies tested. The presence of sensitizing reagents such as cycloheximide and various cytostatic drugs further enhanced antibody-mediated killing of the tumor cells. We believe that these results may point the way to a successful application of bispecific CD95 antibodies in experimental tumor therapy.