Although bispecific antibodies directed against malignant lymphoma have been shown to be effective in vitro and in vivo, extended clinical trials so far have been hampered by the fact that conventional approaches to produce these antibodies suffer from low yields, ill-defined byproducts, or laborious purification procedures. To overcome this problem, we have generated a small, recombinant, lymphoma-directed, bispecific single-chain (bsc) antibody according to a novel technique recently described. The antibody consists of 2 different single-chain Fv fragments joined by a glycine-serine linker. One specificity is directed against the CD3 antigen of human T cells, and the other antigen-binding site engages the pan-B-cell marker CD19, uniformly expressed on the vast majority of B-cell malignancies. The construct was expressed in Chinese hamster ovary cells and purified by its C-terminal histioline tag. Specific binding to CD19 and CD3 was demonstrated by fluorescence-activated cell sorter analysis. By redirecting unstimulated primary human T cells derived from the peripheral blood against CD19-positive lymphoma cells, the bscCD19 x CD3 antibody showed significant cytotoxic activity at very low concentrations of 10 to 100 pg/mL and at effector to target cell ratios as low as 2:1. Moreover, strong lymphoma-directed cytotoxicity at low antibody concentrations was rapidly induced during 4 hours even in experiments without any T-cell prestimulation. Thus, this particular antibody proves to be much more efficacious than the bispecific antibodies described until now. Therefore, the described bscCD19 x CD3 molecule should be a suitable candidate to prove the therapeutic benefit of bispecific antibodies in the treatment of non-Hodgkin lymphoma. (Blood. 2000;95:2098-2103)