A bispecific single-chain antibody directed against EpCAM/CD3 in combination with the cytokines interferon alpha and interleukin-2 efficiently retargets T and CD3(+)CD56(+) natural-killer-like T lymphocytes to EpCAM-expressing tumor cells

Cytokine-induced killer cells (CIK), generated in vitro from peripheral blo od mononuclear cells (PBMC) by addition of interferon gamma (IFN gamma), in terleukin-2 (IL-2), IL-I and a monoclonal antibody (mAb) against CD3, are h ighly efficient cytotoxic effector cells with the CD3(+)CD56(+) phenotype. In this study, we evaluated whether the cytotoxicity of these natural-kille r-like T lymphocytes against the colorectal tumor cell line HT29 can be enh anced by the addition of a bispecific single-chain antibody (bsAb) directed against EpCAM/CD3. For determination of bsAb-redirected cellular cytotoxic ity we used a new flow-cytometric assay, which directly counts viable tumor cells and can assess long-term cytotoxicity. We found that this bsAb induc ed distinct cytotoxicity at a concentration above 100 ng/ml with both PBMC and CIK at an effector-to-target cell ratio as low as 1:1. CIK cells reveal ed higher bsAb-redirected cytotoxicity than PBMC. Cellular cytotoxicity app eared after 24 h whereas PBMC showed the highest bsAb-redirected cytotoxici ty after 72 h. The addition of the cytokines IL-2 and IFN alpha but not gra nulocyte/macrophage-colony-stimulating factor enhanced bsAb-redirected cyto toxicity of both PBMC and CIK. When the bsAb was combined with the murine m Ab BR55-2, which recognizes the Lewis(y) antigen, bsAb-redirected cytotoxic ity was partly augmented, whereas murine mAb 17-1A, which binds to EpCAM as well, slightly suppressed bsAb-redirected cytotoxicity induced by the bsAb . We conclude that CIK generated in vitro or in vivo combined with this new EpCAM/CD3 bsAb and the cytokine IL-2 should be evaluated for the treatment of EpCAM-expressing tumors.